Methods of lowering lipid levels in a mammal

ABSTRACT

This invention relates to methods for lowering lipid levels in mammals using compounds that inhibit advanced glycation endproducts (AGEs), LR-9, LR-74 and LR-90. These compounds, which inhibit non-enzymatic protein glycation, also inhibit the formation of advanced lipoxidation endproducts (ALEs) on target proteins by trapping intermediates in glycoxidation and lopoxidation and inhibiting oxidation reactions important in the formation of AGEs and ALEs.

This application claims the benefit of prior co-pending U.S. ProvisionalApplication Ser. No. 60/514,476, the disclosures of which are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Technical Field

This application relates to the field of biomedical sciences, and inparticular relates to methods for lowering lipid levels in mammals. Someembodiments are directed to inhibition comprising administeringcompounds such as 4-(2-napthylcarboxamido)phenoxyisobutyric acid];2-(8-quinolinoxy) propionic acid]; and methylenebis(4,4′-(2-chlorophenylureidophenoxyisobutyric acid)].

2. Description of the Background Art

The Diabetic Control and Complications Trial (DCCT) and UKPDS studieshave identified hyperglycemia as the main risk factor for thedevelopment of diabetic complications. The Diabetes Control andComplications Trial Research Group N. Engl. J. Med. 329: 977-986, 1993;UK Prospective Diabetes Study Group Lancet 352: 837-853, 1998. Formationof advanced glycation endproducts (AGEs) has been identified as themajor pathogenic link between hyperglycemia and the long-termcomplications of diabetes. Makita et al., N. Eng. J. Med. 325: 836-842,1993; Bucala and Cerami, Adv. Pharmacol 23: 1-33, 1992; Browlee, Nature414: 813-820, 2001; Sheetz and King, J.A.M.A. 288: 2579-2588, 2002;Stith et al., Expert Opin. Invest. Drugs 11: 1205-1223, 2002.

Non-enzymatic glycation (also known as the Maillard reaction) is acomplex series of reactions between reducing sugars and the amino groupsof proteins, lipids, and DNA which leads to browning, fluorescence, andcross-linking. Bucala et al., Proc. Natl. Acad. Sci. USA 90: 6434-6438,1993; Bucala et al., Proc. Natl. Acad. Sci. USA 81: 105-109, 1984; Singhet al., Diabetologia 44: 129-146, 2001. This complex cascade ofcondensations, rearrangements and oxidation produces heterogeneous,irreversible, proteolysis-resistant, antigenic products known asadvanced glycation endproducts (AGEs). Singh et al., Diabetologica 44:129-146, 2001; Ulrich and Cerami, Rec. Prog. Hormone Res. 56: 1-2, 2001.Examples of these AGEs are N^(ε)-(carboxymethyl)lysine (CML),N^(ε)-(carboxyethyl)lysine (CEL), N^(ε)-(carboxymethyl)cysteine (CMC),arg-pyrimidine, pentosidine and the imidazolium crosslinksmethyl-gloxal-lysine dimer (MOLD) and glyoxal-lysine dimer (GOLD).Thorpe and Baynes, Amino Acids 25: 275-281, 2002; Chellan and Nagaraj,Arch. Biochem. Biophys. 368: 98-104, 1999. This type of glycation beginswith the reversible formation of a Schiff's base, which undergoes arearrangement to form a stable Amadori product.

Both Schiff's bases and Amadori products further undergo a series ofreactions through dicarbonyl intermediates to form AGEs. Lipidperoxidation of polyunsaturated fatty acids (PUFA), such as arachidonicacid and linoleic acid, also yield carbonyl compounds. Some of these areidentical to those formed from carbohydrates, such as MG and GO, andothers are characteristic of lipid, such as malondialdehyde (MDA),4-hydroxynonenal (HNE), and 2-hydroxyheptanal (2HH). See Baynes andThorpe, Free Rad. Biol. Med. 28: 1708-1716, 2000; Fu et al., J. Biol.Chem. 271: 9982-9986, 1996; Miyata et al., FEBS Lett. 437: 24-28, 1998;Miyata et al., J. Am. Soc. Nephrol. 11: 1744-1752, 2000; Requena et al.,Nephrol. Dial. Transplant. 11(supp. 5): 48-53, 1996; Esterbauer et al.,Free Radic. Biol. Med. 11: 81-128, 1991; Requena et al., J. Biol. Chem.272: 17473-14779, 1997; Slatter et al., Diabetologia 43: 550-557, 2000.These reactive carbonyl species (RCSs) rapidly react with lysine andarginine residues of proteins, resulting in the formation of advancedlipoxidation endproducts (ALEs) such as N^(ε)-carboxymethyllysine (CML),N^(ε)-carboxyethyllysine (CEL), GOLD, MOLD, malondialdehyde-lysine(MDA-lysine), 4-hydroxynonenal-lysine (4-HNE-lysine), hexanoyl-lysine(Hex-lysine), and 2-hydroxyheptanoyl-lysine (2HH-lysine). See FIG. 1.Thorpe and Baynes, Amino Acids 25: 275-281, 2002; Miyata et al., FEBSLett. 437: 24-28, 1998; Miyata et al., J. Am. Soc. Nephrol. 11:1744-1752, 2000; Uchida et al., Arch. Biochem. Biophys. 346: 45-52,1997; Baynes and Thorpe, Free Rad. Biol. Med. 28: 1708-1716, 2000. SinceCML, CEL, GOLD and MOLD can result from lipid and carbohydratemetabolism, these chemical modifications on tissue proteins that canserve as biomarkers of oxidative stress resulting from sugar and lipidoxidation. Fu et al., J. Biol. Chem. 271: 9982-9986, 1996; Requena etal., Nephrol. Dial. Transplant. 11(supp. 5): 48-53, 1996. The relativerole of hyperglycemia versus hyperlipidemia in the chemical modificationand pathogenesis of diabetic complications remains uncertain, however.Additionally, several biomarkers of protein modification such as CML andCEL can be derived from either sugar or lipid sources, furthercomplicating the interpretation and analysis of experimental data.

In human diabetic patients and in animal models of diabetes, thesenon-enzymatic reactions are accelerated and cause accumulation of AGEson long-lived structural proteins such as collagen, fibronectin,tubulin, lens crytallin, myelin, laminin and actin, in addition tohemoglobin, albumin, LDL-associated proteins and apoprotein. Thestructural and functional integrity of the affected molecules, whichoften have major roles in cellular functions, are perturbed by thesemodifications, with severe consequences on organs such as kidney, eye,nerve, and micro-vascular functions, which consequently leads to variousdiabetic complications such as nephropathy, atherosclerosis,microangiopathy, neuropathy and retinopathy. Boel et al., J. DiabetesComplications 9: 104-129, 1995; Hendrick et al., Diabetologia 43:312-320, 2000; Vlassara and Palace, J. Intern. Med. 251: 87-101, 2002.

Current research indicates that reactive carbonyl species such as MGO,GO, GLA, dehydroascorbate, 3-deoxyglucosone and malondialdehyde, arepotent precursors of AGE/ALE formation and protein crosslinking. Lyonsand Jenkins, Diabetes Rev. 5: 365-391, 1997; Baynes and Thorpe, Diabetes48: 1-9, 1999; Miyata et al., J. Am. Soc. Nephrol. 11: 1744-1752, 2000;Thornalley st al., Biochem. J. 344: 109-116, 1999. In vitro studiesfurther suggest that these carbonyls originate mainly formed fromascorbate and polyunsaturated fatty acids and not from glucose per se.Miyata et al., FEBS Lett. 437: 24-28, 1993.

Direct evidence implicates the contribution of AGEs/ALEs in theprogression of diabetic complications in different lesions of thekidneys, the rat lens, and in atherosclerosis. Horie et al., J. Clin.Invest. 100: 2995-3004, 1997; Matsumoto et al., Biochem. Biophys. Res.Commun. 241: 352-354, 1997; Bucala and Vlassara, Exper. Physiol. 82:327-337, 1997; Bucala and Rahbar, in: Endocrinology of CardiovascularFunction. E. R. Levin and J. L. Nadler (eds.), 1998. Kluwer Acad.Publishers, pp. 159-180; Horie et al., J. Clin. Invest. 100: 2995-3004,1997; Friedman, Nephrol. Dial. Transplant. 14(supp. 3): 1-9, 1999;Kushiro et al., Nephron 79: 458-468, 1998. Several lines of evidenceindicate that hyperglycemia in diabetes causes the increase in reactivecarbonyl species (RCS) such as methylglyoxal, glycolaldehyde, glyoxal,3-deoxyglucosone, malondialdehyde, and hydroxynonenal. “Carbonyl stress”leads to increased modification of proteins and lipids, through reactivecarbonyl intermediates forming adducts with lysine residues of proteins,followed by oxidative stress and tissue damage. Lyons and Jenkins,Diabetes Rev. 5: 365-391, 1997; Baynes and Thorpe, Diabetes 48: 1-9,1999; Miyata et al., J. Am. Soc. Nephrol. 11: 1744-1752, 2000. See FIG.1.

A number of recent clinical trials such as the DCCT/EDIC, EURODIABProspective Complications Study Group, the Hoorn Study and UKPDS, haveunanimously identified plasma trigylceride concentrations as anindependent risk for development of diabetic complications (retinopathy,nephropathy, cardiovascular disease) in diabetic individuals and in thenon-diabetic population. Jenkins et al., Kidney Int. 64: 817-828, 2003;Chaturvedi et al., Kidney Int. 60: 219-227, 2001; van Leiden et al.,Diabetes Care 25: 1320-1325, 2002; United Kingdom Prospective DiabetesStudy (UKPDS: 10), Diabetologia. 36: 1021-1029, 1993. These studies haveestablished a strong correlation between microalbuminuria and levels ofplasma triglycerides and cholesterol. Moreover, recent studies on thelipid-lowering effects of pyridoxamine (PM) and aminoguanidine (AG), twoknown AGE inhibitors in diabetic and hyperlipidemic rats (Degenhardt etal., Kidney Int. 61: 939-950; 2002; Alderson et al., Kidney Int. 63:2123-2133, 2003), suggested that there was increased lipid peroxidationin these animals and that PM and AG in fact had lipid-lowering effects.Furthermore, the lipid lowering effects of PM and the correlation ofplasma triglycerides and cholesterol with AGEs in skin collagensuggested that lipids might be an important source of AGEs in diabeticrats. Several PM adducts of lipoxidation intermediates of arachidonicacid and linoleic acid were excreted in substantially higherconcentrations in the urine of diabetic and hyperlipemic rats treatedwith PM, suggesting an increase in lipoxidation in these animals. Metzet al., J. Biol. Chem. [Aug. 15, Epub ahead of print], 2003. Based onthese results, the authors concluded that lipids could be the primarysource of chemical modification of proteins in diabetes and obesity,especially in the presence of hyperlipidemia or dyslipidemia, even inthe absence of hyperglycemia. Alderson et al., Kidney Int. 63:2123-2133, 2003; Metz et al., J. Biol. Chem. [Aug. 15, Epub ahead ofprint], 2003.

Over the years, several natural and synthetic compounds have beenproposed and advanced as potential AGE/ALE inhibitors. These includeaminoguanidine, pyridoxamine, OPB-9195, carnosine, metformin, as well assome angiotensin-converting enzyme inhibitors (ACEI) and angiotensin IItype 1 receptor blockers (ARB), derivatives of aryl (and heterocyclic)ureido, and aryl (and heterocyclic) carboxamido phenoxyisobutyric acids.Rahbar et al., Biochem. Biophys. Res. Commun. 262: 651-656, 1999; Rahbaret al., Mol. Cell. Biol. Res. Commun. 3: 360-366, 2000; Rahbar andFigarola, Curr. Med. Chem. (Immunol. Endocr. Metabol. Agents) 2:135-161, 2002; Rahbar and Figarola, Curr. Med. Chem. (Immunol. Endocrin.Metabol.) 2: 174-186, 2002; Forbes et al., Diabetes 51: 3274-3282, 2002;Metz et al., Arch. Biochem. Biophys. 419: 41-49; Nangaku et al., J. Am.Soc. Nephrol. 14: 1212-1222, 2003; Rahbar and Figarola, Arch. Biochem.Biophys. 419: 63-79, 2003. Recently, some of these compounds were foundto be effective AGE inhibitors in vivo and to prevent the development ofdiabetic nephropathy in a streptozotocin-induced diabetes.

Over the last decade, evidence has accumulated implicating AGEs/ALEs asmajor factors in the pathogenesis of diabetic nephropathy and othercomplications of diabetes. Administration of AGEs to non-diabetic ratsleads to glomerulosclerosis and albuminuria, indicating that AGEs alonemay be sufficient to cause renal injury in diabetes. Vlassara et al.,Proc. Natl. Acad. Sci. USA 91: 11704-11708, 1994. Diabetic animals fedwith a diet low in glycoxidation products developed minimal symptoms ofdiabetic nephropathy compared with animals fed with diet high inglycoxidation products. Zheng et al., Diabetes Metab. Res. Rev. 18:224-237, 2002. It is widely accepted that AGEs/ALEs contribute todiabetic tissue injury by at least two major mechanisms. Browlee, Nature414: 813-820, 2001; Stith et al., Expert Opin. Invest. Drugs 11:1205-1223, 2002; Vlassara and Palace, J. Intern. Med. 251: 87-101, 2002.The first is receptor-independent alterations of the extracellularmatrix architecture and function of intracellular proteins by AGE/ALEformation and AGE/ALE-protein crosslinking. The other isreceptor-dependent modulation of cellular functions through interactionof AGE with various cell surface receptors, especially RAGE. Wendt etal., Am. J. Pathol. 162: 1123-1137, 2003; Vlassara, Diabetes Metab. Res.Rev. 17: 436-443, 2001; Kislinger et al., J. Biol. Chem. 274:31740-3174, 1999.

Advanced glycation/lipoxidation endproducts (AGEs/ALEs) also have beenimplicated in the pathogenesis of a variety of debilitating diseasessuch as atherosclerosis, Alzheimer's and rheumatoid arthritis, as wellas the normal aging process. The pathogenic process is accelerated whenelevated concentrations of reducing sugars or lipid peroxidationproducts are present in the blood and in the intracellular environmentsuch as occurs with diabetes. Both the structural and functionalintegrity of the affected molecules become perturbed by thesemodifications and can result in severe consequences in the short andlong term. Because hyperlipidemia, hyperglycemia, diabetes and syndromessuch as “metabolic syndrome” are common and are a common cause ofmorbidity and mortality, methods to counteract the symptoms andconsequences of these metabolic states are needed in the art.

SUMMARY OF THE INVENTION

Accordingly, in one embodiment the invention provides a method oflowering lipid levels in a mammal comprising administering to the mammalan effective amount of of any of the following compounds orpharmaceutically acceptable salts thereof: LR-9[4-(2-napthylcarboxamido) phenoxyisobutyric acid]; LR-74[2-(8-quinolinoxy)propionic acid]; and LR-90 [methylenebis(4,4′-(2-chlorophenylureidophenoxyisobutyric acid)].

In another embodiment, the invention provides a method of treatingcomplications resulting from diabetes which result from elevated levelsof lipids, the method comprising administering an effective amount ofany of the following compounds or pharmaceutically acceptable saltsthereof: LR-9 [4-(2-napthylcarboxamido)phenoxyisobutyric acid]; LR-74[2-(8-quinolinoxy)propionic acid]; and LR-90 [methylene bis(4,4′-(2-chlorophenylureidophenoxyisobutyric acid)].

In yet another embodiment, the invention provides a method of treating apatient with Menkes Disease, Wilson's Disease, or X-linked Cutis Laxa,which comprises administering an effective amount of of any of thefollowing compounds or pharmaceutically acceptable salts thereof: LR-9[4-(2-napthylcarboxamido)phenoxyisobutyric acid]; LR-74[2-(8-quinolinoxy)propionic acid]; and LR-90 [methylenebis(4,4′-(2-chlorophenylureidophenoxyisobutyric acid)].

In in vivo studies investigating the effects of three compounds (LR-9,LR-74 and LR-90) in streptozotocin-induced diabetic rats, the compoundsof the present invention not only were able to inhibit the process ofAGE formation and prevent early renal disease, but also inhibited theformation of advanced lipoxidation endproducts (ALEs) duringlipoxidation reactions and efficiently reduced the increasedconcentrations of triglycerides and cholesterol in diabetic animals bymore than 50%, preventing the complications normally seen in diabetesand in aging.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing metabolic sources of reactive carbonylspecies and carbonyl stress. Asterisks in the diagram represent thepostulated pathways where the LR compounds exert their effects.

FIG. 2 shows the chemical structures of compounds LR-9[4-(2-napthylcarboxamido)phenoxyisobutyric acid]; LR-74[2-(8-quinolinoxy)propionic acid]; and LR-90 [methylene bis(4,4′-(2-chlorophenylureidophenoxyisobutyric acid)].

FIG. 3 shows total serum triglycerides (3B) and cholesterol (3A),measured in non-diabetic animals (ND), diabetic animals (D) and diabeticanimals treated with LR-90 for 32 weeks (* indicates p<0.05 vs.non-diabetic control; **=p<0.05 vs. diabetic control).

FIG. 4 shows total plasma triglycerides (4A) and cholesterol (4B),measured in non-diabetic animals (ND), diabetic animals (D) and diabeticanimals treated with LR-9 or LR-74 for 30 weeks (* indicates p<0.05 vs.non-diabetic control; **=p<0.05 vs. diabetic control).

FIG. 5 shows the effect of LR compounds on serum AGE after LR-90treatment for 32 weeks. Serum AGE concentration was measuredimmunologically using anti-AGE RNAse polyclonal antibodies. *=p<0.05 vs.non-diabetic control; **p<0.05 vs. diabetic control.

FIG. 6 shows the effect of LR compounds on serum AGE after LR-9 or LR-74treatment for 30 weeks. Serum AGE concentration was measuredimmunologically using anti-AGE RNAse polyclonal antibodies. *=p<0.05 vs.non-diabetic control; **p<0.05 vs. diabetic control.

FIG. 7 shows the effect of LR compounds on rat tail tendon crosslinkingmeasured by pepsin digestion (fluorescence). * indicates p<0.05 vs.non-diabetic control; ** indicates p<0.05 vs. diabetic control.

FIG. 8 shows the effect of LR compounds on rat tail tendon crosslinkingmeasured by solubility in weak acid. * indicates p<0.05 vs. non-diabeticcontrol; ** indicates p<0.05 vs. diabetic control.

FIG. 9 Shows the effect of LR-90 on the degree of glomerulosclerosis indiabetic rats. Glomerulosclerotic index (GSI) was calculated from 150glomeruli in each rat. * indicates p<0.05 vs. non-diabetic control; **indicates p<0.05 vs. diabetic control.

FIG. 10 is a series of photographs showing LR-90 reduced basementmembrane thickening. Kidney sections were cut and photographed usinghigh resolution TEM to show basement membrane expansion and thickening.

FIG. 11 is series of photographs of trichrome-stained kidney sectionsshowing collagen deposition and cortical tubule degeneration in kidneysfrom non-diabetic, diabetic and diabetic rats treated with LR-90.Formalin-fixed kidney sections rats from each treatment group at 32weeks were mounted on slides and stained with trichrome. (A)non-diabetic; (B), diabetic; (C) diabetic+LR-90.

FIG. 12 is a series of photographs of picrosirius red-stained kidneysections showing collagen deposition in kidney. Formalin-fixed kidneysections rats from each treatment group at 32 weeks were mounted onslides and stained with Picrosirius red. (A) non-diabetic; (B),diabetic; (C) diabetic+LR-90.

FIG. 13 shows immunohistochemical staining for AGE. Formalin-fixedkidney sections of rats from each treatment group at 32 weeks weremounted on slides and stained with 6D12 monoclonal anti-AGE antibodiesspecific for CML. (A) non-diabetic; (B), diabetic; (C) diabetic+LR-90.

FIG. 14 shows immunohistochemical staining for nitrotyrosine.Formalin-fixed kidney sections of rats from each treatment group at 32weeks were mounted on slides and stained anti-nitrotyrosine polyclonalantibodies. (A) non-diabetic; (B), diabetic; (C) diabetic+LR-90.

FIG. 15 provides data on inhibition of Cu⁺⁺-catalyzed oxidation ofascorbic acid by LR compounds compared with known AGE inhibitors. Thedashed horizontal line indicates 50% loss of AA.

FIG. 16 provides data for inhibition of Cu⁺⁺-mediated lipid peroxidationby LR compounds.

FIG. 17 shows the effect of LR compounds on free radical production.Hydroxyl radicals were measured from the hydroxylation of benzoate byH₂O₂ and expressed as salicylate equivalents obtained from salicylicacid standards (17A). Superoxide production was monitored from thereaction with methylgloxal with N-α-acetyl-L-lysine and detected by theWST-1 assay (17B).

FIG. 18 shows the effects of diabetes and LR treatment on renal CML-AGEaccumulation. Magnification 200×. 18A: ND; 18B: D; 18C: D+LR-9; 18D:D+LR-74.

FIG. 19 shows the effects of diabetes and LR treatment on levels offluorescent AGE in tail tendon collagen. (A) Tail tendon collagen wasdigested with pepsin and the supernatant analyzed for fluorescent AGEand OH-proline content. (* indicates p<0.01 vs. ND, ** indicates p<0.05vs. D).

FIG. 20 shows the effects of diabetes and LR treatment on levels ofAGEs/ALEs in skin collagen. Skin collagen was analyzed forconcentrations of CML (20A) and CEL (20B) using LC-ESI/MS/MS. *indicates p<0.01 vs. ND; ** indicates p<0.05 vs. D.

FIG. 21 shows the effects of diabetes and LR treatment on theconcentration of (A) plasma lipids and (B) plasma lipid hydroperoxidesin STZ-diabetic rats. * indicates p<0.01 vs. ND; ** indicates p<0.05 vs.D; indicates p<0.01 vs. D.

FIG. 22 is a series of photographs showing the effect of diabetes and LRcompounds on renal protein oxidation. 22A: non-diabetic control; 22B:diabetic control; 22C: diabetic plus LR-9; 22D: diabetic plus LR-74.Magnification is 200×.

FIG. 23 shows inhibition of Cu⁺⁺-mediated LDL oxidation by LR compounds.

FIG. 24 shows the effects of LR compounds on the kinetics of linoleicacid oxidation. The MDA equivalent was estimated based on standards.Values are means±SD of two independent experiments with n=4 pertreatment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The LR compounds discussed here (see FIG. 2) belong to a group of novelaromatic compounds derived from LR-16. LR-16 acts as an allostericeffector, synergistic with 2,3-bisphosphoglycerate in increasing theoxygen affinity of hemoglobin molecules, and which has been shown tolower serum cholesterol and low-density lipoproteins (LDL) in rats whichwere fed a diet rich in cholesterol. Lalezari et al., Proc. Natl. Acad.Sci. USA 85: 6117-6121, 1988.

The studies presented here showed that diabetic rats treated with any ofthe new LR compounds provided statistically significant improvement inrenal function in terms of development of proteinuria and reduction increatinine excretion. In addition, histochemical observations showedthat treatment with these LR compounds minimized kidney structuraldamage as indicated by a reduction in the incidence ofglomerulosclerosis, cortical tubule degeneration and collagen depositionin the kidney compared to untreated diabetic rats. Additionally, thecompounds prevented mesangial expansion and basement membrane thickeningof the kidneys of diabetic rats. These compounds effectively inhibitedthe increase in serum AGE and the in situ accumulation of immunoreactiveAGEs in collagen tissues and kidneys of diabetic rats without any effecton hyperglycemia. The LR compounds lowered cholesterol and triglycerideconcentrations found in the hyperlipidemia of diabetic rats but did notsignificantly change in the lipid levels of control non-diabetic rats.

Without wishing to be bound by theory, two proposed mechanisms for thebeneficial effects of the LR compounds in preventing diabeticnephropathy, are their lipid lowering activities per se, or their AGEinhibitor and antioxidative properties. Reduction of plasma lipids bytreatment with lipid lowering compounds such as statins has been shownto provide protection against nephropathy in non-diabetic obese rats.O'Donnell et al., Am. J. Kidney Dis. 22: 83-89, 1993; Oda and Keane,Kidney Int. Suppl. 71: S2-S5, 1999. On the other hand, the AGE/ALEinhibitor pyridoxamine have been also shown to correct hyperlipidemiaand nephropathy in both diabetic rats and non-diabetic rats obese rats,probably by interfering with various reactive carbonyl intermediates ofAGE/ALE formation from lipid oxidation. Degenhardt et al., Kidney Int.61: 939-950, 2002; Alderson et al., Kidney Int. 63: 2123-2133, 2003.Unlike pyridoxamine, which has minimal effects on lipid peroxidation,all three LR compounds were strong inhibitors of LDL oxidation in vitro.

Thus, in addition to its protective effects on kidneys in diabetic rats,these novel compounds can be used in the treatment of atherosclerosisand other vascular complications of diabetes. Such additional beneficialeffects were unexpected. Although there was the possibility that theincreaseed lipid peroxidation seen in the diabetic rats is correlatedwith the higher substrate levels (increased plasma lipid levels) inthese animals relative to non-diabetic rats, there was no significantcorrelation between plasma cholesterol or triglyceride concentration andthe levels of plasma lipid hydroperoxides in both non-diabetic anddiabetic control rats. These results suggest that lipid peroxidation maybe independent of the total available lipids in the plasma, which isconsistent with earlier observations in human and animal studies.Griesmacher et al., Am. J. Med. 98: 469-475, 1995; Ihm et al.,Metabolism 48: 1141-1145, 1999. More importantly, these findingsindicate that the elevated lipid peroxidation products could beassociated with increased oxidative stress as a consequence of increasedAGE/ALE formation in the diabetic rats.

Known AGE inhibitors with renoprotective effects such aminoguanidine,pyridoxamine, and OPB-9195 are thought to prevent AGE/ALE accumulationby interacting with highly reactive RCS and acting as carbonyl traps,preventing AGE/ALE formation. However, the metal chelation properties ofthese AGE inhibitors may contribute to their effectiveness in preventingAGE formation in vivo. The mechanism of action of these LR compounds isstill unclear, but the LR compounds are potent chelators of Cu²⁺ (morepotent than AG and PM), and are effective inhibitors of oxidation ofascorbic acid. Moreover, these compounds strongly inhibit hydroxylradical formation, and LR-90 also may prevent superoxide production. Thevarious pathways involved in the production and generation of proteincarbonyls and Amadori products important in the formation of some AGEsand ALEs may require free radicals, transition metals, or both. Miyataet al., J. Am. So. Nephrol. 13: 2478-2487, 2002; Voziyan et al., J.Biol. Chem. (2003 Sep. 15) [Epub ahead of print]. However, unlikeaminoguanidine and pyridoxamine which act primarily by trapping RCS,these novel LR compounds also reduce the production of RCS byinterfering with oxidative metabolism, for example by lowering formationof hydroxyl radicals and interacting with metal ions that can furtherpromote sugar/lipid oxidation reactions.

Notably, the compounds LR-9, -74 and -90 are potent inhibitors of thecopper catalyzed oxidation of ascorbic acid. This observation points toseveral additional uses of LR-9, -74 or -90, including as therapeuticsin conditions, syndromes or diseases involving copper. In the body,copper ions can be found in the cuprous (Cu⁺) or the cupric (Cu⁺⁺)states. In general, diseases involving copper fall into two maincategories: (1) diseases involving environmental exposure to copper,including excess levels of copper and (2) diseases involving coppermetabolism, the distribution of copper within the body and the role ofcopper in biological processes, including the involvement of copper inenzymes or biological processes. Some copper-related enzymes implicatedin disease include: superoxide dismutase (Cu/Zn SOD) (implicated inamyothrophic lateral sclerosis); tyrosine hydroxylase anddopamine-beta-hydroxylase that form or catabolize several brainneurotransmitters such as dopamine and norepinephrine; monoamine oxidase(MAO) which plays a role in the metabolism of the neurotransmittersnorepinephrine, epinephrine, and dopamine and also functions in thedegradation of the neurotransmitter serotonin; lysyl oxidase which isrequired for the cross-linking of collagen and elastin; and cytochrome coxidase which is involved in the synthesis of phospholipids whichcomprise structures such as the myelin sheath of neurons.

Ceruloplasmin, a copper binding protein, is thought to prevent freecopper ions from catalyzing oxidative damage. Ceruloplasmin hasferroxidase activity (oxidation of ferrous iron) which facilitates ironloading onto its transport protein. This transfer may prevent freeferrous ions (Fe²⁺) from promoting the generation of free radicals.Thus, the level of serum copper and/or the copper-loading status ofceruloplasmin may modulate iron metabolism. Copper-dependenttranscription factors may affect the transcription of specific genesincluding genes for Cu/Zn SOD, catalase (another antioxidant enzyme),and proteins related to the cellular storage of copper. Further specificdisease conditions involving copper metabolism include Menkes Disease(also known as Menkes Kinky Hair Syndrome), Wilson's Disease andX-linked Cutis Laxa (also known as type IX Ehlers-Danlos syndrome orOccipital Horn syndrome) listed via OMIN reference numbers #309400,#277900, and #304150 respectively. The particular symptoms seen in theseconditions (for example, osteoporosis, neurodegeneration in the graymatter of the brain damaged cerebral arteries leading to vascularrupture or blockage in Menkes patient) may indicate more general rolesfor copper in such symptoms or disease processes such as cerebrovascularinfarction, vascular rupture, etc.

Thus, copper plays a broad role in a number of biological pathwayspresent in normal or disease states where therapeutic intervention usingcompounds that modulate copper would be advantageous. The desirableactivities of such compounds may include, but may not be limited tochelation of free copper in solution, mobilization of copper fromcarrier proteins, optimal distribution of copper into the preferredbiological compartment, the optimal sequestration of copper intobiological compartments and/or the promotion of copper excretion.

The effective dosages and modes of administration are made in accordancewith accepted medical practices taking into account the clinicalcondition of the individual subject (e.g. severity and course of thedisease), the site and method of administration, scheduling ofadministration, patient age, sex, body weight and other factors known tomedical practitioners. Accordingly, the dosages of the compositions ofthe invention for treatment of a subject are to be titrated to theindividual subject. For example, the interrelationship of dosages foranimals of various sizes and species and humans based on mg/m² ofsurface area is described by Freireich et al., Cancer Chemother. Rep.50(4): 219-244 (1966). The “effective dose” can be determined byprocedures known in the art, and must be such as to achieve adiscernible change in the disease state.

In addition to their effects on AGE formation and lipid metabolism, LRtreatment also may influence some steps in the inflammation pathwaysleading to tissue injury. LR-90 also prevented cell infiltration in therenal interstitium of diabetic rats. In fact, no neutrophils weredetected in LR-90 treated diabetic rats, which were numerous and indense aggregates in untreated diabetic rats, important because CMLformation at the site of tissue injury is promoted by enzymaticcatalysis by neutrophils. Activated neutrophils usemyeloperoxidase-hydrogen peroxide-chloride system to converthydroxy-amino acids into GLA and other reactive aldehydes which areprecursors for CML. Anderson et al., J. Clin. Invest. 104: 103-113,1999. Such in vivo production of CML precursors could play a major rolein the renal pathology observed here by generating additional AGEproduction at the site of injury since CML adducts of proteins areligands for AGE that activate cell signaling pathway and modulate geneexpression. In vitro and in vivo studies indicate that CML and other AGEcan enhance formation of reactive oxygen species and induce NF-κBactivation in proximal endothelial cells, perpetuating an increase inproinflammatory gene products, cytokines, adhesion molecules, and ROSthat all can contribute to renal damage. Morcos et al., Diabetes 51:3532-3544, 2002; Boulanger et al., Kidney Int. 61: 148-156, 2002; Bastaet al., Circulation 105: 816-822, 2002.

LR-90 treatment decreased the overall oxidative damage to renal tissues,as shown by nitrotyrosine formation in renal cortex. Recent studiesindicate that increased nitrotyrosine concentrations play a major rolein early diabetic tubular damage and in the progression of renaldisease. Thuraisingham et al., Kidney Int. 57: 968-972, 2002. Proximaltubular cells produce nitric oxide (NO), which can react with superoxideto form peroxynitrite (ONOO⁻), a powerful oxidant. Peroxynitritenitrosylates tyrosine moieties on proteins, producing nitrotyrosine.Beckman and Koppenol, Am. J. Physiol. 271(5 Pt 1): C1424-C1437, 1996;Reiter et al., J. Biol. Chem. 275: 32460-32466, 2000. In vitro studieshave suggested that glycation itself can result in the production ofsuperoxide and hydroxyl radicals through transition metal. Sakurai etal., FEBS Lett. 236: 406-410, 1988; Yim et al., J. Biol. Chem. 270:28228-28233, 1995; Ortwerth et al., Biochem. Biophys. Res. Commun. 245:161-165, 1998. The increase in CML-AGE and nitrotyrosine staining inrats with diabetic nephropathy can be attenuated by ramipril andaminoguanidine, indicating that ACE inhibition and blockage of AGEformation could involve common pathways such as ROS formation. Forbes etal., Diabetes 51: 3274-3282, 2002.

AGE/ALE formation can stimulate the oxidation of lipids by generation ofoxidizing intermediates, including free radicals, in the presence oftrace amounts of iron or copper which act as catalysts. Formation offree radicals is enhanced in diabetes by glucose oxidation(glycoxidation), non-enzymatic glycation of proteins, oxidativedecomposition of glycated proteins, and interaction of AGEs/ALEs withRAGE. Abnormally high levels of free radicals and the simultaneousdecline in antioxidant defense mechanisms may lead to increasedoxidative stress and subsequent lipid peroxidation. As shown in thepresent study, diabetic animals exhibited higher levels of oxidativestress in both kidneys and plasma as indicated by enhanced nitrotyrosinestaining in the kidney tubules and glomeruli, and increased lipidhydroperoxides in plasma. Evidence in both experimental and clinicalstudies suggests that hyperglycaemia-induced oxidative stress can play amajor role in the lipid metabolism in diabetes.

Both glucose oxidation and glycation can catalyze PUFA peroxidation ofcell membranes. In high glucose-environment, proteins and lipoproteinstrapped within tissues can undergo glycation to produce ROS and lipidperoxidation products. However, based on the results of this studyuntreated and LR-treated diabetic rats showed no difference in glucoseand HbAlc concentration, indicating that hyperglycaemia alone might haveonly limited influence the levels of lipid peroxidation. In contrast,there were significant differences in the levels of AGE/ALE in collagenand in kidneys of untreated and LR-treated diabetic animals, concomitantwith decreased concentrations of lipids and lipid peroxidation productsafter LR treatment. Taken together, these data suggest that inhibitionof AGEs/ALEs formation can prevent oxidative stress and subsequentdamage in diabetic animals.

Recent studies with the AGE/ALE inhibitor PM in normoglycemic Zuckerobese and hyperlipemic rats have raised some interesting questions aboutwhether lipids and ALEs, and not carbohydrates and AGEs, are responsiblefor most of chemical modifications and tissue damage in diabetes Mert etal., J. Biol. Chem. 278: 42012-42019, 2003; Januszewski et al., Biochem.Soc. Trans. B1: 1413-1415, 2003; Alderson et al., Kidney Int. 63:2123-2133, 2003. Oxidative stress in these rats can trigger the onset ofkidney lesions and renal dysfunction, concurrent with the firstappearance of lipid peroxidation products and decline of antioxidantenzyme activities. See Poirier et al., Nephrol. Dial. Transplant. 15:467-476, 2000. Overall, these studies suggest that hyperlipidaemia andlipid peroxidation can independently induce renal impairment in theabsence of hyperglycaemia. Additionally, both hypercholesterolemia andhypertriglyceridaemia are recognized as independent risk factors for thedevelopment of renal disease and are also associated with nephroticsyndromes independent of diabetes. Furthermore reduction of plasmalipids by lipid lowering drugs (e.g., statins) have successfullyresulted in renoprotection against diabetic nephropathy. As documentedin this study, LR-9 and LR-74 inhibit lipid peroxidation reactions, andtherefore possess general antioxidant properties. Both these compoundshave weaker carbonyl trapping activities compared with AG, PM and LR-90,but are strong inhibitors of hydroxyl radicals formation, andconsequently may be working on a different AGE/ALE inhibition mechanismcompared with these prototype AGE inhibitors.

Oxygen, redox active transition metals and ROS are catalysts of AGE andALE formation. The various pathways involved in the production andgeneration of RCS and Amadori products, important in the formation ofsome AGEs and ALEs, thus may require free radicals, transition metals,or both. However, unlike AG and PM, which act primarily by trapping RCS,the LR compounds discussed here also may reduce the product of RCS byinterfering with oxidative metabolism, probably by inhibiting formationof free radicals and interacting with metal ions that can furtherpromote sugar/lipid oxidation reactions. Here, LR compounds reduced thelevels of AGEs/ALEs such as CML and CEL, inhibited the chemicalmodifications of collagens, and decreased the overall oxidative stressin plasma and kidneys of diabetic animals. All these effects caninfluence the thickening and loss of elasticity of the vascular wall,membrane permeability, and inflammatory process (via RAGE interaction),which can lead to the prevention of dyslipidaemia.

Regardless of how the LR compounds lower plasma lipids and inhibit lipidperoxidation reactions in vivo, such effects further broaden thepossible therapeutic applications of these compounds. Decomposition oflipid peroxides initiates chain of reactions that produce various RCSthat can generate AGEs and ALEs and various lipid adducts which can leadto the accumulation of lipids and lipoproteins in form cells in vascularwall. LDL has been identified as the major carrier of lipidhydroperoxides in the plasma and oxidative modification of LDL has beensuggested as a causal step in the development of atherosclerosis.Redox-active transition metals and fee radicals, as well as AGEformation and glycoxidation, have been implicated in this process. Whilethere is conflicting evidence for the actual involvement of transitionmetals in modifying LDL in vivo, human atherosclerotic lesions containelevated levels of redox-active copper and iron, and variousantioxidants have been shown to inhibit LDL oxidation and retard thedevelopment of atherosclerosis in human and animal models. Metalchelation therapy is effective in improving endothelial function inpatients with coronary artery disease. Thus agents that can inhibitAGE/ALE formation and reduce the oxidative stress are in a position toprevent the development of atherosclerosis in diabetic subjects. Theability of LR compounds to chelate copper in this study could be one ofthe mechanisms for the observed inhibition of lipid peroxidationreactions in vitro and in vivo, since no adducts formed between thefatty acid (linoleic acid) and the compounds were detected usingRP-HPLC, and these compounds were not consumed in the lipoxidationreaction. In addition, neither compound had any effect oflipoxygenase-mediated LDL oxidation. These findings further reinforcethe showing that these LP compounds inhibit AGE/ALE formation, and atleast to some extent lipid peroxidation reactions, mainly through theirantioxidant/metal chelation properties. The overall superiorrenoprotective, lipid-lowering and anti-lipid peroxidation effects ofLR-74 relative to LR-9 in the present study could be a reflection of thebetter antioxidant and hydroxyl radical scavenger characteristics of theformer compound Figarola et al., Diabetologia 46: 1140-1152. However,while both drugs were given at 50 mg/L, LR-74 was administered at about1½ times the LR-9 dose (about 0.15 mmol/L for LR-9 vs. 0.23 mmol/L forLR-74). Assuming similar bioavailability and pharmacokinetics, theeffects of LR-9 are as impressive as LR-74 despite these differences indosages administered in animals.

In summary, we have identified compounds that can inhibit AGE/ALEformation in vivo and also delay or inhibit the progression of earlyrenal dysfunction in diabetic animals. These compounds also preventhyperlipidaemia and inhibit the overall oxidative stress in theseanimals. The LR compounds described here can be an effective treatmentmodality for early renal disease and other diabetic complications whereaccumulation of AGEs/ALEs and intermediate compounds are primarycontributors. Aside from their AGE inhibitory properties, thesecompounds possess lipid-lowering characteristics that can influence boththe development of diabetic renal disease and atherosclerosis. Theability of the compounds to chelate transition metals, their interactionwith RCS and/or intervention with RCS formation, as well as inhibitingfree radical production, could be mediating the renoprotective andlipid-lowering effects of these compounds.

REFERENCES

-   1. Abrass, Cellular lipid metabolism and the role of lipids in    progressive renal disease. Am. J. Nephrol. 24: 46-53, 2004.-   2. Al-Abed et al., Advanced glycation end products: detection and    reversal. Methods Enzymol. 309: 152-172, 1999.-   3. Alderson et al., The AGE inhibitor pyridoxamine inhibits lipemia    and development of renal and vascular disease in Zucker obese rats.    Kidney Int. 63: 2123-2133, 2003.-   4. Altomare et al., Increased lipid peroxidation in type 2 poorly    controlled diabetic patients. Diabetes Metab. 18: 264-271, 1992.-   5. Anderson et al., The myeloperoxidase system of human phagocytes    generates N-epsilon(carboxymethyl)lysine on proteins: a mechanism    for producing advanced glycation end products at sites of    inflammation. J. Clin. Invest. 104: 103-113, 1999.-   6. Basta et al., Advanced glycation end products activate    endothelium through signal-transduction receptor RAGE: a mechanism    for amplification of inflammatory responses. Circulation 105:    816-822, 2002.-   7. Baynes and Thorpe, Perspective in diabetes: role of oxidative    stress in diabetes complications. A new perspective on an old    paradigm. Diabetes 48: 1-9, 1999.-   8. Baynes and Thorpe, Glycoxidation and lipoxidation in    atherogenesis. Free Rad. Biol. Med. 28: 1708-1716, 2000.-   9. Beckman and Koppenol, Nitric oxide, superoxide, and    peroxynitrite: the good, the bad, and ugly. Am. J. Physiol. 271(5 Pt    1): C1424-C1437, 1996.-   10. Boel et al., Diabetic late complications: will aldose reductase    inhibitors or inhibitors of advanced glycosylation endproduct    formation hold promise? J. Diabetes Complications 9: 104-129, 1995.-   11. Boulanger et al., AGEs bind to mesothelial cells via RAGE and    stimulate VCAM-1 expression. Kidney Int. 61: 148-156, 2002.-   12. Browlee, Biochemistry and molecular cell biology of diabetic    complications. Nature 414: 813-820, 2001.-   13. Bucala et al., Modification of DNA by reducing sugars: a    possible mechanism for nucleic acid aging and age-related    dysfunction in gene expression. Proc. Natl. Acad. Sci. USA 81:    105-109, 1984.-   14. Bucala and Cerami, Advanced glycosylation: chemistry, biology    and implications for diabetes and aging. Adv. Pharmacol. 23: 1-33,    1992.-   15. Bucala et al., Lipid advanced glycation pathway for lipid    oxidation. Proc. Natl. Acad. Sci. USA 90: 6434-6438, 1993.-   16. Bucala and Vlassara, Lipid and lipoprotein modification by    advanced glycation end-products: Role in atherosclerosis. Exper.    Physiol. 82: 327-337, 1997.-   17. Bucala and Rahbar, Protein glycation and vascular disease. In:    Endocrinology of Cardiovascular Function. E. R. Levin and J. L.    Nadler (eds.). (1998) Kluwer Acad. Publishers, pp. 159-180.-   18. Carew et al., Antiatherogenic effect of probucol unrelated to    its hypocholesterolemic effect: evidence that antioxidants in vivo    can selectively inhibit low density lipoprotein degradation in    macrophage-rich fatty streaks and slow the progression of    atherosclerosis in the Watanabe heritable hyperlipidemic rabbit.    Proc. Natl. Acad. Sci. USA 84: 7725-7729, 1987.-   19. Carpenter et al., Oral alpha-tocopherol supplementation inhibits    lipid oxidation in established human atherosclerotic lesions. Free    Radic. Res. 37: 1235-1244, 2003.-   20. Chaturvedi et al., Microalbuminuria in type 1 diabetes: rates,    risk factors and glycemic threshold. Kidney Int. 60: 219-227, 2001.-   21. Chellan and Nagaraj, Protein crosslinking by the Maillard    reaction: dicarbonyl-derived imidazolium crosslinks in aging and    diabetes. Arch. Biochem. Biophys. 368: 98-104, 1999.-   22. Chung et al., Single vertical spin density gradient    ultracentrifugation. Methods Enzymol. 128: 181-209, 1978.-   23. Creemers et al., Microassay for the assessment of low levels of    hydroxyproline. Biotechniques 22: 656-658, 1997.-   24. Degenhardt et al., Pyridoxamine inhibits early renal disease and    dyslipidemia in the streptozotocin-diabetic rat. Kidney Int. 61:    939-950, 2002.-   25. The Diabetes Control and Complications Trial Research Group    (1993). The effect of intensive treatment of diabetes on the    development and progression of long-term complications in    insulin-dependent diabetes mellitus. N. Engl. J. Med. 329: 977-986,    1993.-   26. Dillon et al., Antioxidant properties of aged garlic extract: an    in vitro study incorporating human low density lipoprotein. Life    Sci. 72: 1583-1594, 2003.-   27. Duffy et al., Iron chelation improves endothelial function in    patients with coronary artery disease. Circulation 103: 2799-2804,    2001.-   28. Esterbauer et al., Chemistry and biochemistry of    4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic.    Biol. Med. 11: 81-128, 1991.-   29. Figarola et al., LR-90, a new advanced glycation endproduct    inhibitor prevents progression of diabetic nephropathy in    STZ-diabetic rats. Diabetologia 46: 1140-1152, 2003.-   30. Forbes et al., Reduction of the accumulation of advanced    glycation end products by ACE inhibition in experimental diabetic    nephropathy. Diabetes 51: 3274-3282, 2002.-   31. Friedman, Advanced glycation end-products in diabetic    nephropathy. Nephrol. Dial. Transplant. 14(Suppl 3): 1-9, 1999.-   32. Fu et al., The advanced glycation end product,    N^(ε)-(carboxymethyl) lysine, is a product of both lipid    peroxidation and glycoxidation reactions. J. Biol. Chem. 271:    9982-9986, 1996.-   33. Giardino et al., Aminoguanidine inhibits reactive oxygen species    formation, lipid peroxidation, and oxidant-induced apoptosis.    Diabetes 47: 1114-1120, 1998.-   34. Gogasyavuz et al., Effects of aminoguanidine on lipid and    protein oxidation in diabeic rat kidneys. Int. J. Exp. Diabetes Res.    3: 145-151, 2002.-   35. Griesmacher et al., Enhanced serum levels of    thiobarbituric-acid-reactive substances in diabetes. Am. J. Med. 98:    469-475, 1995.-   36. Heinecke, Oxidants and antioxidants in the pathogenesis of    atherosclerosis: implications for the oxidized low density    lipoprotein hypothesis. Atherosclerosis 141: 1-15, 1998.-   37. Hendrick et al., Glycation impairs high-density lipoprotein    function. Diabetologia 43: 312-320, 2000.-   38. Hicks et al., Catalysis of lipid peroxidation by glucose and    glycosylated collagen. Biochem. Biophys. Res. Commun. 151: 649-655,    1988.-   39. Horie et al., Immunohistochemical colocalization of glyoxidation    products and lipid peroxidation products in diabetic renal    glomerular lesions. J. Clin. Invest. 100: 2995-3004, 1997.-   40. Ihm et al., Effect of aminoguanidine on lipid peroxidation in    streptozotocin-induced diabetic rats. Metabolism 48: 1141-1145,    1999.-   41. Inouye et al., Glycated hemoglobin and lipid peroxidation in    erythrocytes of diabetic patients. Metabolism 48: 205-209, 1999.-   43. Jain et al., Erythrocyte membrane lipid peroxidation and    glycosylated hemoglobin in diabetes. Diabetes 38: 1539-1543, 1989.-   44. Januszewski et al., Role of lipids in chemical modification of    proteins and development of complications in diabetes. Biochem. Soc.    Trans. 31: 1413-1416, 2003.-   45. Jenkins et al., Lipoproteins in the DCCT/EDIC cohort:    associations with diabetic nephropathy. Kidney Int. 64: 817-828,    2003.-   46. Joles et al., Early mechanisms of renal injury in    hypercholesterolemic or hypertriglyceridemic rat. J. Am. Soc.    Nephrol. 11: 669-683, 2000.-   47. Kawamura et al., Pathophysiological concentrations of glucose    promotes oxidative modification of low density lipoprotein by a    superoxide-dependent pathway. J. Clin. Invest. 942: 771-778, 1994.-   48. Kennedy and Lyons, Glycation, oxidation, and lipoxidation in the    development of diabetic complications. Metabolism 46: 14-21, 1997.-   49. Kislinger et al., N(epsilon)-(carboxymethyl)lysine adducts of    proteins are ligands for receptor for advanced glycation end    products that activate cell signaling pathways and modulate gene    expression. J. Biol. Chem. 274: 31740-3174, 1999.-   50. Knott et al., Glycation and glycoxidation of low-density    lipoproteins by glucose and low-molecular mass aldehydes. Formation    of modified and oxidized particles. Eur. J. Biochem. 270: 3572-3582,    2003.-   51. Kochakian et al., Chronic dosing with aminoguanidine and novel    advanced glycosylation end product-formation inhibitors ameliorates    cross-linking of tail tendon collagen in STZ-induced diabetic rats.    Diabetes 45: 1694-1700, 1996.-   52. Kushiro et al., Accumulation of N sigma-(carboxymethyl)lysine    and changes in glomerular extracellular matrix components in Otsuka    Long-Evans Tokushima fatty rat: a model of spontaneous NIDDM.    Nephron 79: 458-468, 1998.-   53. Lalezari et al., LR16, a compound with potent effects on the    oxygen affinity of hemoglobin, on blood cholesterol, and on low    density lipoprotein. Proc. Natl. Acad. Sci. USA 85: 6117-6121, 1988.-   54. Lam et al., Cholesterol-lowering therapy may retard the    progression of diabetic nephropathy. Diabetologia 38: 604-609, 1995.-   55. Lamb et al., Transistion metal ions within human atherosclerotic    lesions can catalyse the oxidation of low-density lipoprotein by    macrophages. FEBS Lett. 374: 12-16, 1995.-   56. Lo et al., The reaction of methylglyoxal with aminoguanidine    under physiological conditions and prevention of methylglyoxal    binding to plasma proteins. Biochem. Pharmacol. 48: 1865-1870, 1994.-   57. Lopes-Virella et al., Modification of lipoprotein in diabetes.    Diabetes Metab. Rev. 12: 69-90, 1996.-   58. Lyons et al., Glycation, oxidation and lipoxidation in the    development of the complications of diabetes mellitus: a ‘carbonyl    stress’ hypothesis. Diabetes Rev. 5: 365-391, 1997.-   59. Makita et al., Advanced glycosylation end products in patients    with diabetic nephropathy. N. Eng. J. Med. 325: 836-842, 1993.-   60. Matsumoto et al., Immunohistochemical evidence for increased    formation of advanced glycation end products and inhibition by    aminoguanidine in diabetic rat lines. Biochem. Biophys. Res. Commun.    241: 352-354, 1997.-   61. Metz et al., Pyridoxamine, an inhibitor of advanced glycation    and lipoxidation reactions: a novel therapy for treatment of    diabetic complications. Arch. Biochem. Biophys. 419: 41-49.-   62. Metz et al., Pyridoxamine traps intermediates in lipid    peroxidation reactions in vivo: evidence on the role of lipids in    chemical modification of protein and development of diabetic    complications. J. Biol. Chem. 278: 42012-42019, 2003.-   63. Miyata et al., Generation of protein carbonyls by glycoxidation    and lipoxidation reactions with autoxidation products of ascorbic    acid and polyunsaturated fatty acids. FEBS Lett. 437: 24-28, 1993.-   64. Miyata et al., Advanced glycation and lipoxidation end products:    role of reactive carbonyl compounds generated during carbohydrate    and lipid metabolism. J. Am. Soc. Nephrol. 11: 1744-1752, 2000.-   65. Miyata et al., Angiotensin II receptor antagonists and    angiotensin-converting enzyme inhibitors lower in vitro the    formation of advanced glycation end products: biochemical    mechanisms. J. Am. So. Nephrol. 13: 2478-2487, 2002.-   66. Miyata et al., Angiotensin II receptor blockers and angiotensin    converting enzyme inhibitors: implication of radical scavenging and    transition metal chelation in inhibition of advanced glycation end    product formation. Arch. Biochem. Biophys. 419: 50-54, 2003.-   67. Morcos et al., Activation of tubular epithelial cells in    diabetic nephropathy. Diabetes 51: 3532-3544, 2002.-   68. Mowri et al., Glucose enhancement of LDL oxidation is strictly    metal ion dependent. Free Radic. Biol. Med. 29: 814-824, 2000.-   69. Mullarkey et al., Free radical generation by early glycation    products: a mechanism for accelerated atherogenesis in diabetes.    Biochem. Biophys. Res. Commun. 173: 771-778, 1994.-   70. Muntner et al., Plasma lipids and risk of developing renal    dysfunction: the atherosclerosis risk in communities study. Kidney    Int. 58: 293-301, 2000.-   71. Nagaraj et al., Effects of pyridoxamine on chemical    modifications of proteins by carbonyls in diabetic rats:    characterization of a major product from the reaction of    pyridoxamine with methylglyoxal. Arch. Biochem. Biophys. 402:    110-119, 2002.-   72. Nourooz-Zadeh et al., Low-density lipoprotein is the major    carrier of lipid hydroperoxides in plasma. Relevance to    determination of total plasma lipid hydroperoxide concentrations.    Biochem. J. 313: 781-786, 1996.-   73. Nangaku et al., Anti-hypertensive agents inhibit in vivo the    formation of advanced glycation end products and improve renal    damage in a type 2 diabetic nephropathy rat model. J. Am. Soc.    Nephrol. 14: 1212-1222, 2003.-   74. Oda and Keane, Recent advances in statins and the kidney. Kidney    Int. Suppl. 71: S2-S5, 1999.-   75. O'Donnell et al., Lovastatin retards the progression of    established glomerular disease in obese Zucker rats. Am. J. Kidney    Dis. 22: 83-89, 1993.-   76. Ortwerth et al., The generation of superoxide anions in    glycation reactions with sugars, osones, and 3-deoxyosones. Biochem.    Biophys. Res. Commun. 245: 161-165, 1998.-   77. Poirier et al., Oxidative stress occurs in absence of    hyperglycaemia and inflammation in the onset of kidney lesions in    normotensive obese rats. Nephrol. Dial. Transplant. 15: 467-476,    2000.-   78. Price et al., Chelating activity of advanced glycation    end-products inhibitors. J. Biol. Chem. 276: 48967-48972, 2001.-   79. Rahbar et al., Novel inhibitors of glycation endproducts.    Biochem. Biophys. Res. Commun. 262: 651-656, 1999.-   80. Rahbar et al., Novel inhibitors of advanced glycation    endproducts (Part II). Mol. Cell. Biol. Res. Comm. 3: 360-366, 2000.-   81. Rahbar and Figarola, Inhibitors and breakers of advanced    glycation endproducts (AGEs): a review. Curr. Med. Chem.—Immunol.    Endocr. Metabol. Agents 2: 135-161, 2002.-   82. Rahbar and Figarola, Inhibitors and breakers of advanced    glycation endproducts. Curr. Med. Chem.—Immunol. Endocrin. Metabol.    2: 174-186, 2002.-   83. Rahbar and Figarola, Novel inhibitors of advanced glycation    endproducts. Arch. Biochem. Biophys. 419: 63-79, 2003.-   84. Reiter et al., Superoxide reacts with nitric oxide to nitrate    tyrosine at physiological pH via peroxynitrite. J. Biol. Chem. 275:    32460-32466, 2000.-   85. Requena et al., Lipoxidation products as biomarkers of oxidative    damage to proteins during lipid peroxidation reactions. Nephrol.    Dial. Transplant. 11(Suppl 5): 48-53, 1996.-   86. Requena et al., Carboxymethylethanolamine: a biomarker of    phospholipid modification during the Maillard reaction in vivo. J.    Biol. Chem. 272: 17473-14779, 1997.-   87. Sakata et al., Glycoxidation and lipid peroxidation of    low-density lipoprotein can synergistically enhance atherogenesis.    Cardiovasc. Res. 49: 466-475, 2001.-   88. Sakurai and Tsuchiya, Superoxide production from    nonenzymatically glycated protein. FEBS Lett. 236: 406-410, 1988.-   89. Satoh, Serum lipid peroxide in cerebrovascular disorders    determined by a new colorimetric method. Clin. Chim. Acta 90: 37-43,    1978.-   90. Shaw et al., N-epsilon-(carboxymethyl)lysine (CML) as a    biomarker of oxidative stress in long-lived tissue proteins. Methods    Mol. Biol. 186: 129-137, 2002.-   91. Sheetz and King, Molecular understanding of hyperglycemia's    adverse effects for diabetic complications. JAMA 288: 2579-2588,    2002.-   92. Singh et al., Advanced glycation end-products: a review.    Diabetologia 44: 129-146, 2001.-   93. Slatter et al., The importance of lipid-derived malondialdehyde    in diabetes mellitus. Diabetologia 43: 550-557, 2000.-   94. Smith et al., Stimulation of lipid peroxidation and    hydroxyl-radical generation by the contents of human atherosclerotic    lesions. Biochem. J. 286: 901-905, 1992.-   95. Stadler et al., Direct detection and quanification of transition    metal ions in human atherosclerotic plaques: evidence for the    presence of elevated levels of iron and copper. Arterioscler.    Thromb. Vasc. Biol. 24: 949-954, 2004.-   96. Stefek et al., p-Dimethyl aminobenzaldehyde-reactive substances    in tail tendon collagen of streptozotocin-diabetic rats: temporal    relation to biomechanical properties and advanced glycation    endproduct (AGE)-related fluorescence. Biochim. Biophys. Acta 1502:    398-404, 2000.-   97. Stith et al., Advanced glycation end products and diabetic    complications. Expert Opin. Invest. Drugs 11: 1205-1223, 2002.-   98. Teuscher et al., Nephropathy subsequent to hyperlipidemia. Clin.    Nephrol. 54: 64-67, 2000.-   99. Thornalley et al., Formation of glyoxal, methylglyoxal and    3-deoxyglucosone in the glycation of proteins by glucose.    Biochem. J. 344: 109-116, 1999.-   100. Thornalley et al., Kinetics and mechanism of the reaction of    aminoguanidine with the alpha-oxoaldehydes glyoxal, methylglyoxal,    and 3-deoxyglucosone under physiological conditions. Biochem.    Pharmacol. 60: 55-65, 2000.-   101. Thorpe and Baynes, Role of oxidative stress in development of    complications in diabetes: a new perspective on an old paradigm.    Diabetes 48: 1-9, 1999.-   102. Thorpe and Baynes, Maillard reaction products in tissue    proteins: new products and new perspectives. Amino Acids 25:    275-281, 2002.-   103. Thuraisingham et al., Increased nitrotyrosine staining in    kidneys from patients with diabetic nephropathy. Kidney Int. 57:    968-972, 2000.-   104. Uchida et al., Protein modification by lipid peroxidation    products: formation of malondialdehyde-derived    N(epsilon)-(2-propenol)lysine in proteins. Arch. Biochem. Biophys.    346: 45-52, 1997.-   105. Ukeda et al., Spectrophotometric assay of superoxide anion    formed in Maillard reaction based on highly water-soluble    tetrazolium salt. Anal. Sci. 18: 1151-1154, 2002.-   106. Ulrich and Cerami, Protein glycation, diabetes & aging. Recent    Prog. Horm. Res. 56: 1-21, 2001.-   107. United Kingdom Prospective Diabetes Study (UKPDS: 10) (1993)    Urinary albumin excretion over 3 years in diet-treated Type 2,    (non-insulin dependent) diabetic patients, and association with    hypertension, hyperglycemia and hypertriglyceridaemia. Diabetologia    36: 1021-1029, 2003.-   108. United Kingdom Prospective Diabetes Study Group (1998).    Intensive blood-glucose control with sulphonylureas or insulin    compared with conventional treatment and risk of complications in    patients with type 2 diabetes (UKPDS 33). Lancet 352: 837-853, 1998.-   109. van Leiden et al., Blood pressure, lipids, and obesity are    associated with retinopathy: the Hoorn Study. Diabetes Care 25:    1320-1325, 2002.-   110. Vlassara et al., Advanced glycation end-products induce    glomerular sclerosis and albuminuria in normal rats. Proc. Natl.    Acad. Sci. USA 91: 11704-11708, 1994.-   111. Vlassara, The AGE-receptor in the pathogenesis of diabetic    complications. Diabetes Metab. Res. Rev. 17: 436-443, 2001.-   112. Vlassara and Palace, Diabetes and advanced glycation    endproducts. J. Intern. Med. 251: 87-101, 2002.-   113. Voziyan et al., A post-Amadori inhibitor pyridoxamine also    inhibits chemical modification of proteins by scavenging carbonyl    intermediates of carbohydrate and lipid degradation. J. Biol. Chem.    277: 3397-3403, 2002.-   114. Voziyan et al., Modification of proteins In vitro by    physiological levels of glucose: Pyridoxamine inhibits conversion of    amadori intermediate to advanced glycation end-products through    binding of redox metal ions. J. Biol. Chem. 2003 Sep. 15 [Epub ahead    of print].-   115. Wautier et al., Activation of NADPH oxidase by AGE links    oxidant stress to altered gene expression via RAGE. Am. J. Physiol.    Endocrinol. Metab. 280: E685-E694, 2001.-   116. Wendt et al., RAGE drives the development of glomerulosclerosis    and implicates podocyte activation in the pathogenesis of diabetic    nephropathy. Am. J. Pathol. 162: 1123-1137, 2003.-   117. Wilkinson-Berka et al., ALT-946 and aminoguanidine, inhibitors    of advanced glycation, improve severe nephropathy in the diabetic    transgenic (mREN-2) 27 rat. Diabetes 51: 3283-3289, 2002.-   118. Wolff, Diabetes mellitus and free radicals. Free radicals,    transistion metals and oxidative stress in the aetiology of diabetes    mellitus and complications. Br. Med. Bull. 49: 642-652, 1993.-   119. Yan et al., Glycation, inflammation, and RAGE: a scaffold for    the macrovascular complications of diabetes and beyond. Circ. Res.    93: 1159-1169.-   120. Yang et al., AGE-breakers cleave model compounds, but do not    break Maillard crosslinks in skin and tail collagen from diabetic    rats. Arch. Biochem. Biophys. 412: 42-46, 2003.-   121. Yim et al., Free radicals generated during the glycation    reaction of amino acids by methylglyoxal. A model study of    protein-cross-linked free radicals. J. Biol. Chem. 270: 28228-28233,    1995.-   122. Zheng et al., Prevention of diabetic nephropathy in mice by a    diet low in glycoxidation products. Diabetes Metab. Res. Rev. 18:    224-237, 2002.

EXAMPLES Example 1 Treatment of Diabetic and Control Rats

Male Sprague-Dawley rats (about 175 to 200 g) were adapted for one weekprior to treatment, then rendered diabetic by intra-peritoneal injectionof STZ (65 mg/kg in citrate buffer, pH 4.5) after an overnight fast.Control (non-diabetic) animals were injected with the buffer only.Diabetes was confirmed by measuring the plasma glucose concentrations 7days after Streptozotocin (STZ)-injection. Only animals with a plasmaglucose concentration greater than 20 mmol were classified as diabeticand used in the study. These diabetic rats were divided randomly into anuntreated diabetic control group and a diabetic treatment group. Thetreatment group received an LR compound at 50 mg/l in their drinkingwater throughout the duration of the study (32 weeks for LR-90; 30 weeksfor LR-9 and LR-74). All animals were housed individually and were givenfree access to food and water.

Blood (from the tail vein) and urine samples were collected from ratsfor glycemic control analysis and albuminuria measurements. Glycemia wasmonitored every 8 weeks by measuring plasma glucose and glycatedhemoglobin. Plasma or serum also was tested for total cholesterol andtotal triglycerides. Progression of renal dysfunction was assessed bymeasuring urinary albumin to creatinine ratio (UA/Cr) and serum orplasma creatinine. For measurement of urinary albumin and creatinineconcentrations, rats were housed in metabolic cages for 24 hours andurine was collected in a collection beaker with several drops of tolueneto inhibit microbial growth.

At the end of study, the rats were weighed and anaesthetized withisoflourane and blood was drawn by heart puncture and transferred intoheparinized and non-heparinized vacutainer tubes on ice. These bloodsamples were later centifuged for plasma and serum collectionrespectively, and stored at −70° C. until the time of analysis. Ratswere killed by over-anesthetization and cardiac puncture and the kidneyswere removed immediately, weighed, decapsulated and rinsed in PBSbuffer. Sections of the kidneys were stored in 10% NBF for subsequentmicroscopic examinations and immunohistochemistry. The tail of eachindividual rat was cut, removed and stored in 50 mL conical tubes at−70° C.

Diabetic rats had significantly increased plasma glucose and glycatedhemoglobin concentrations compared with control rats (p<0.01). SeeTables I and II. Diabetes was also associated with reduced weight gain.Treatment of diabetic rats with compounds LR-9, LR-74 and LR-90 did notaffect plasma glucose and glycated hemoglobin, but did result in amoderate increase in weight compared to the diabetic control rats (withonly the LR-90 treatment showing statistical significance). Severaldiabetic rats treated with the LR compounds did not reach the end of thestudy period, but the incidence was not increased compared with thediabetic controls. Additionally, no mortality was recorded fromnon-diabetic control rats and those non-diabetic rats treated with allthe LR compounds.

Statistical analyses of data presented in this example and the followingexamples were first analyzed by ANOVA and post-hoc comparisons betweengroup means were analyzed using unpaired Student's t test. A p value ofless than 0.05 was considered statistically significant. Data arepresented as means±SD. TABLE I Effects of LR-90 on Body Weight andGlycemia in STZ- Diabetic Rats (32 Week Treatment). Body Weight PlasmaGlucose Group n (g) (mmol/l) HbA1c (%) ND^(a) 4 691.0 ± 94.1  7.5 ± 1.01.4 ± 0.1 ND + LR-90 4 723.0 ± 21.9  6.8 ± 0.7 1.5 ± 0.1 D^(a) 5 267.2 ±71.3 31.3 ± 5.3^(b) 3.8 ± 0.2^(b) D + LR-90 6 337.6 ± 26.6^(c) 28.9 ±3.4 3.3 ± 0.4^(a)ND = non-diabetic; D = diabetic^(b)p < 0.05 vs. non-diabetic control rats^(c)p < 0.05 vs. diabetic rats

TABLE II Effects of LR-9 and LR-74 on Body Weight and Glycemia inSTZ-Diabetic Rats (30 Week Treatment). Body Weight Plasma Glucose Groupn (g) (mmol/l) HbA1c (%) ND^(a) 4 668.5 ± 65.5  8.5 ± 0.7 0.90 ± 0.08ND + LR-9 4 681.0 ± 9.3  8.2 ± 0.9 0.90 ± 0.08 ND + LR-74 4 744.0 ± 51.8 6.8 ± 0.8 0.87 ± 0.12 D^(a) 5 250.8 ± 43.3^(b) 26.5 ± 2.3^(b) 2.06 ±0.09 D + LR-9 6 288.0 ± 71.8 27.1 ± 1.3 1.95 ± 0.22^(b) D + LR-74 6314.7 ± 53.0 26.9 ± 1.5 2.10 ± 0.30^(a)ND = non-diabetic; D = diabetic^(b)p < 0.05 vs. non-diabetic control rats^(c)p < 0.05 vs. diabetic rats

Example 2 Effects on Lipid Metabolism

Diabetic rats showed elevated levels of both total plasma/serumtriglycerides and cholesterol compared with non-diabetic rats (p<0.001).See FIG. 3. Diabetic rats treated with any of the LR compounds showedsignificant reduction in both triglyceride and cholesterolconcentrations. LR-90 reduced serum triglycerides and serum cholesterolby 50% towards levels of non-diabetic animals (FIG. 3). Similarly,plasma triglycerides and cholesterol levels of diabetic rats were alsoreduced by more than 60% and 50%, respectively by both LR-9 and LR-74(FIG. 4).

Example 3 Effects on Renal Function

Urinary albumin, plasma creatinine concentration, and urinaryalbumin/creatine ratio (UA/Cr) were used as indicators of renalfunction. Compared with non-diabetic control rats, urinary albuminexcretion, plasma creatine concentration and UA/Cr increasedsignificantly in diabetic animals. See Tables III and IV. Treatment ofdiabetic rats with the LR compounds inhibited the rise in urinaryalbumin excretion and UA/Cr, with about a 50% reduction in concentrationcompared to untreated diabetics rats. In addition, the elevated plasmacreatinine concentrations observed in diabetic animals weresignificantly decreased by almost 50% with treatment of either LR-9,LR-74 or LR-90. TABLE III Effects of LR-90 on Renal Function Parametersin STZ-diabetic Rats. Urinary Plasma Urinary Albumin/ Creatine AlbuminCreatine Group (mg/dl) (mg/24 hr) Ratio ND^(a) 0.46 ± 0.07  7.6 ± 0.80.49 ± 0.24 ND + LR-90 0.58 ± 0.07  8.9 ± 1.5 0.57 ± 0.16 D^(a) 2.94 ±0.90^(b) 37.5 ± 8.4^(b) 3.32 ± 1.37^(b) D + LR-90 1.50 ± 0.53^(c) 23.6 ±4.5^(c) 1.57 ± 0.49^(c)^(a)ND = non-diabetic; D = diabetic^(b)p < 0.05 vs. non-diabetic control rats^(c)p < 0.05 vs. diabetic rats

TABLE IV Effects of LR-9 and LR-74 on Renal Function Parameters inSTZ-diabetic Rats. Urinary Plasma Urinary Albumin/ Creatine AlbuminCreatine Group (mg/dl) (mg/24 hr) Ratio ND^(a) 0.45 ± 0.11  4.8 ± 1.80.34 ± 0.10 ND + LR-9 0.42 ± 0.02  4.8 ± 2.3 0.33 ± 0.13 ND + LR-74 0.42± 0.05  4.6 ± 2.4 0.30 ± 0.16 D^(a) 3.13 ± 0.85^(b) 32.8 ± 8.0^(b) 2.91± 0.85^(b) D + LR-9 1.79 ± 0.95^(c) 18.0 ± 9.1^(c) 1.36 ± 0.93^(c) D +LR-74 1.64 ± 1.07^(c) 14.3 ± 9.07^(c) 1.23 ± 0.60^(c)^(a)ND = non-diabetic; D = diabetic^(b)p < 0.05 vs. non-diabetic control rats^(c)p < 0.05 vs. diabetic rats

Example 4 Effects on Serum AGE

Serum AGE was measured according to the methods of Al-Abed et al., Meth.Enzymol 309: 152-172, 1999 and quantitated with polyclonal R6/9 anti-AGERNAse antibodies using the methods of Rahbar et al., Biochem. Biophys.Res. Commun. 262: 651-656, 1999. One AU was assumed equivalent to 1μg/ml AGE-BSA.

Diabetic rats had about five-fold increase in levels of serum AGEcompared to non-diabetic rats (p<0.05). See FIGS. 5 and 6. Diabetic ratstreated with the LR compounds markedly reduced the AGE concentration byas much as 50%.

Example 5 Collagen Crosslinking, Fluorescence, and Acid Solubility

Isolation and preparation of tail tendon collagen was performedaccording to Kochakian et al., Diabetes 45: 1694-1700, 1996. Therelative degree of crosslinking and AGE formation in collagen wasassessed by pepsin digestion and acid solubility. Pepsin digestion wasperformed as described previously by Stefek et al., Biochim. Biophys.Acta 1502: 398-404, 2000. Briefly, collagen samples of 10 mg fromindividual rats were digested with pepsin (50 μg/mL in 0.5 mol/L aceticacid) for 24 hours at 37° C. After digestion, the samples werecentrifuged at 3000 rpm for 30 minutes at 4° C. and the clearsupernatant containing the digested collagen was collected. One hundredmicroliter aliquots of the supernatant were mixed with 900 μL PBS bufferfor measurement of the fluorescence of the sample at 365 nm excitationand 418 nm emission. The hydroxyproline content of the supernatant wascalculated following acid hydrolysis using a microassay method accordingto known methods (Creemers et al., Biotechniques 22: 656-658, 1997).

The acid solubility of tail tendon collagen was measured by amodification of the method outlined in Yang et al., Arch. Biochem.Biophys. 412: 42-46, 2003. Briefly, samples of about 2 mg collagen wereweighed and solubilized in 0.05 M acetic acid overnight at 4° C. Thesuspensions were centrifuged at 20,000 g 60 minutes at 4° C. and thesupernatants and pellets were separated for analysis of hydroxyprolinecontent following acid hydrolysis. Acid solubility was calculated as thepercentage of hydroxyproline in the supernatant divided by the totalhydroxyproline content in the pellet and the supernatant.

Levels of fluorescent AGE in tail collagen were increased about fourfoldin diabetic rats compared to non-diabetic animals. LR-treated diabeticrats showed a significant reduction of fluorescence and crosslinkingcompared with the untreated diabetic animals (FIG. 7A: LR-90; FIG. 7B:LR-9 and LR-74). Similarly, when tail tendon collagen was solubilized inweak acetic acid, the collagen of diabetic rats showed very lowsolubility in the acid solution. Diabetic rats that received the LRcompounds significantly increase the acid solubility of the tail tendoncollagen (FIG. 8).

Example 6 LR Compound Effects on Kidney Anatomy and Histopathology

To quantitate glomerulosclerosis (defined as glomerular basementmembrane thickening, mesangial hypertrophy, and capillary occlusion),kidney sections were stained with periodic acid Schiff (PAS) reagent. Atotal of 150 glomeruli were randomly chosen from each rat kidney (fourdifferent kidneys per treatment) and carefully graded for sclerosis, bya blinded evaluator. The degree of sclerosis in each glomerulus wasgraded subjectively on a scale of 1 to 4 as follows: grade 1, scleroticarea less than 25%; grade 2, sclerotic area 25-50%; grade 3, scleroticarea 51-75%; and grade 4, sclerotic area more than 75%. Theglomerulosclerotic index (GSI) then was calculated using the followingformula: GSI=Σ⁴ _(i=1) Fi (i), where Fi is the percentage of glomeruliin the rat with a given score of (I). See Wilkinson-Berka et al.,Diabetes 51: 3283-3289, 2002. To quantitate glomerulosclerosis, kidneysections were stained with periodic acid Schiff (PAS) reagent.

Cellular infiltrates were identified in the renal interstitium from 5μm-thick kidney sections stained with PAS. Infiltrates in each kidneysample were graded as follows: + (patchy and light), ++ (patchy anddense), +++ (diffuse and dense with aggregates of neutrophils in tubulesor in interstitium) also in a blinded manner. For collagen depositionstaining in the kidneys, paraffin sections were randomly chosen fromkidneys from each treatment group and stained with Masson's trichrome.Briefly, the sections were deparaffined, hydrated with water, andimmersed in Mordant in Boiuin's solution for 10 minutes. The sectionsthen were rinsed in water and stained with Mayer's hematoxylin for 6minutes. After rinsing in water, Biebrich scarlet-acid fuchsin was addedfor 2 minutes, rinsed, phosphomolybdic-phosphotungstic solution addedfor 15 minutes, followed by aniline blue solution for 10 minutes. Afterrinsing the sections with water, glacial acetic acid was added for 20seconds and then the slides were dehydrated with 95% ethanol. With thismethod, blue and red colors indicate collagen and cytoplasm staining,respectively. Degenerated tubules were identified by the absence ofcytoplasm. Additional staining for collagen fiber deposition in theglomeruli was performed using Picrosirius red staining.

For examination of renal morphometry, kidney samples from each groupwere post-fixed with 2% glutaraldehyde overnight in cacodylate buffer.Sections were cut to 1 μm thickness and stained with Toluidine Blue.Then 80 nm sections were cut with a diamond knife, picked up onformvar-coated, carbon-coated slot copper grids and stained with 5%aqueous uranyl acetate for 15 minutes, followed by 2 minutes incubationon a drop of lead citrate. The grids were observed and photographed witha high resolution transmission electron microscope. The images were usedto determine the width of the glomerular basement membrane and mesangialexpansion.

Mean kidney weights, both in absolute weight and as a fraction of totalbody weight, were significantly increased in diabetic rats compared tonon-diabetic animals, but there were no statistically significantdifferences detected between kidney weight of LR-treated and untreateddiabetic control rats. Occasional cysts were observed in kidneys ofdiabetic control animals, but these were not more frequent in LR-treatedrats. Moreover, there was no evidence of tumor growths in other majororgans (heart, liver, intestines) from both untreated and LR-treateddiabetic animals.

No considerable ultrastructural abnormalities in kidneys were detectedfrom the non-diabetic rats, except for few thickened basement membranesand a few cases of glomerulosclerosis (FIG. 9). Most of the glomerulishowed a normal ultrastructural appearance with normal cellularity, anormal mesangium, and a basement membrane of about 150 nm as revealed byTEM (FIG. 10). No cellular infiltrates were detected in the renalinterstitium from these animals. In untreated diabetic rats, TEM dataindicated that many glomeruli showed thickened basement membranes (about270 nm) with markedly increased cellularity and increased mesangialcells and matrix (FIG. 10), and this was reflected by an increased GSI(FIG. 9). Also, there were a number of cellular infiltrates observed,including dense aggregates of lymphocytes and neutrophils (data notshown).

Diabetic rats treated with the LR compounds also showed an increase incellularity, although less markedly than that of untreated diabeticanimals. Only lymphocytes were observed in the renal interstitium. Inaddition, there was less glomerular damage, thinner basement membrane(about 220 nm), and significantly lower GSI than untreated diabetic rats(p<0.05, FIGS. 9 and 10). Moreover, both collagen deposition (bluecolor) in the tubulointerstitium and glomeruli, and the number ofdegenerate tubules (identified by the absence of cytoplasm or reddishcolor), were increased in diabetic rats compared with the non-diabeticcontrols rats, and LR treatment reduced the amount of collagen stainingand frequency of degenerate tubules to an almost similar extent to thatof the non-diabetic control rats (FIG. 11). Similar results wereobserved when the kidneys were stained with Picrosirius red: LR-90treatment reduced the amount of collagen deposited inside the glomeruliand the tubulointerstitium (FIG. 12).

Example 7 AGE Immunohistochemistry

For immunohistochemical AGE staining, formalin-fixed parafilm embeddedsections (2 μm thick) were mounted on slides coated with2-aminopropyltriethoxy silane, baked for 3 hours at 58° C.,deparaffinized, rinsed with 3% hydrogen peroxide, and incubated withProteinase K (0.5 mg/mL) for 5 minutes at room temperature. Thesesections were washed with rinse buffer and blocked with Protein BlockingAgent for 5 minutes and subsequently incubated with 6D12 anti-AGE mousemonoclonal antibody specific for CML for 30 minutes at room temperature.After washing with rinse buffer, the sections were incubated withEnvision™ with labeled polymer peroxidase-conjugated mouse anti-IgG for30 minutes at room temperature, followed by detection with3,3′-diaminobenzidine tetrahydrochoride solution as chromogen and 50%hematoxylin as counterstain.

Immunohistochemical staining for AGE in rat kidney showed that there waswidespread staining for AGE in the kidney glomeruli and the corticaltubules in diabetic rats compared with the non-diabetic control rats.LR-90 treatment visibly reduced the AGE deposited in these regions (FIG.13). Similar reduction of AGE staining was observed on kidneys of ratstreated with LR-9 or LR-74.

Example 8 Nitrotyrosine Staining

Nitrotyrosine, a marker for protein oxidation, was used as an index ofoxidative tissue damage caused by reactive nitrogen species.Immunohistochemical detection of nitrotyrosine was performed as reportedpreviously (Forbes et al., Diabetes 51: 3274-3282, 2002) and followed inthis study with little modification. Briefly, formalin-fixed kidneysections (5 μm thick), taken from representative rats from eachtreatment group at 32 weeks, were mounted on slides, dewaxed andhydrated. After incubation with Proteinase K for 10 minutes, sectionswere incubated in 3% hydrogen peroxide for 20 minutes, blocked withnormal porcine serum for 20 minutes, and then stained with commerciallyavailable rabbit polyclonal anti-nitrotyrosine antibodies for 1 hour.After rinsing with DAKO rinse buffer, sections were incubated withbiotinylated anti-rabbit IgG for 25 minutes, followed by incubation withavidin-biotin horseradish peroxidase complex for 25 minutes.Localization of peroxidase conjugates was revealed usingdiaminobenzidine tetrahydrochloride (DAB) solution as chromogen and 50%hematoxylin as counterstain.

Nitrotyrosine was predominantly detected in the renal tubules and littlestaining was visible in glomeruli. See FIG. 14. Increased nitrotyrosinestaining was observed in the renal tubules of diabetic rats comparedwith non-diabetic animals, and rats treated with either of the LRcompounds showed markedly reduced nitrotyrosine staining in the corticaltubules. See FIG. 14.

Example 9 In Vitro Tests

In vitro measurement of the kinetics of inhibition of copper-catalyzedoxidation of ascorbic acid was performed according to the methods ofPrice et al., J. Biol. Chem. 276: 48967-48972, 2001. Briefly, CuCl₂ andvarious concentrations of inhibitor compounds were pre-incubated inchelex-treated 20 mmol/L phosphate buffer, pH 7.4, for 5 minutes.Ascorbic acid then was added (50 μL of 10 mmol/L in water) to initiatethe reaction (1 mL total reaction volume). The final concentrations ofCuCl₂ and ascorbic acid in the reaction were 500 nmol/L and 500 μmol/L,respectively. Aliquots (135 μL) were removed at 0 and 60 minutes andtransferred to autoinjector vials containing 15 μL of 10 mmol/l DTPA.Samples were analyzed by reversed phase HPLC on an XTerra™ RP18 column(250 mm×4.6 mm, 5 μm) with an XTerra™ RP18 5 μm guard column using aWaters® 2690 Separator Module equipped with auto-injector and Millenium®32 software. Solvents and gradient were all used as described in Dillonet al., Life Sci. 72: 1583-1594, 2003. The absorbance of ascorbic acidwas measured at 244 nm and the peak area was obtained to estimate thepercent of ascorbic acid remaining versus time. For each inhibitorcompound, the concentration that inhibited the rate of AA oxidation by50% (IC₅₀), was calculated with respect to the control using Prism™software.

The Cu²⁺ chelating activity of the three LR compounds, aminoguanidine(AG) and pyridoxamine (PM) are shown in FIG. 15. In this assay, the IC₅₀values of LR-9, LR-74, LR-90, PM and AG were 200, 50, 275, 1250 and 2750μM, respectively. These results indicate that in vitro, LR-74 was themost potent metal chelator among the LR compounds, and all these novelcompounds were better metal chelators than both known AGE inhibitors AGand PM.

The ability of the LR compounds to inhibit lipid peroxidation was testedusing Cu⁺⁺-mediated lipid oxidation, a common in vitro model for studieson lipoxidative modifications of proteins. The effects of the LRcompounds on lipid peroxidation were studied using Cu⁺⁺-mediatedoxidation of LDL. Human LDL (50 μg of protein/mL) was incubated at 37°C. in chelex-treated PBS buffer alone or in the presence of 5 μM CuCl₂or 5 μM CuCl₂ plus various concentrations of the inhibitor compounds(10-250 μM). After 5 hours of incubation, the amount of thiobarbituricacid reactive substances (expressed as malondialdehyde (MDA)equivalents) generated in the reaction mixture was calculated accordingto methods described by Dillon et al., Life Sci. 72: 1583-1594, 2003.Briefly, aliquots from each sample were precipitated with 20%trichloroacetic acid, centrifuged and an equal volume of 1%thiobarbituric acid was added to the supernatant. The samples then wereheated to 95° C. for 10 minutes, and upon cooling, the absorbance wasread at 532 nm. Hydrolyzed tetraethoxypropane was used as a standard forthe MDA equivalent calculation.

As shown in FIG. 16, all the LR compounds inhibited LDL oxidation in aconcentration-dependent manner. The inhibition activities of LR-74 andLR-90 were better than AG. PM had no effect on lipid peroxidation.

The effects of the compounds on free radical production were evaluatedin a cell-free system. In vitro hydroxyl radical production wasdetermined by the hydroxylation of benzoate by H₂O₂ as described inGiardino et al., Diabetes 47: 1114-1120, 1998. In brief, 30 mmol/Lsodium benzoate in PBS buffer, pH 7.4, was incubated with 10 mmol/L H₂O₂overnight at 37° C. alone and in the presence of various amounts ofinhibitor compounds. After incubation, aliquots from each sample wereanalyzed for fluorescence (305 nm excitation; 408 nm emission). Resultswere expressed as the amount of salicylate equivalent (μM) produced bythe hydroxylation of benzoate. Mannitol, a known hydroxyl radicalscavenger, was included in the experiment as control.

The superoxide radical scavenging activity of the compounds wasevaluated using the WST-1 method described in Ukeda et al., Anal. Sci.18: 1151-1154, 2002. Briefly, methylglyoxal, was incubated with orwithout N-α-acetyl-lysine in 0.05 M chelex-treated sodium phosphatebuffer, pH 7.4, in the presence of various concentrations of theinhibitor compounds. The production of superoxide was monitoredspectrophotometrically at 438 nm, and compared with superoxide dismutaseand Tiron, two known superoxide radical scavengers.

All three LR compounds inhibited ⁻OH radicals formed from the reactionof hydrogen peroxide with sodium benzoate in a concentration-dependentmanner, with greater inhibitory activities than mannitol, a well-known⁻OH radical scavenger. See FIG. 17A. Using the WST-1 assay to monitorsuperoxide produced from an actual glycation reaction, only LR-90 at >1mM showed significant effect on superoxide produced from this reaction.See FIG. 17B. LR-74, as well as the AGE inhibitor aminoguanidine, hadlittle or no effect on superoxide production.

Example 10 LR Compound Treatment of Diabetic Rats

Diabetes was induced in male Sprague-Dawley rats by a single i.p.injection of STZ (65 mg/kg in citrate buffer, pH 4.5) after an overnightfast. Non-diabetic animals were injected with citrate buffer only. Oneweek after STZ injection, only animals with >20 mmol/L plasma glucosewere classified as diabetic and included in the study. Diabetic ratswere divided randomLy into the following treatment groups: diabeticuntreated (D); and two diabetic treatment groups, receiving either LR-9(D+LR-9) or LR-74 (D+LR-74) at 50 mg/L in drinking water. Threenon-diabetic groups were studied concurrently: one untreatednon-diabetic group (ND), and two non-diabetic groups treated with eitherLR-9 (ND+LR-9) or LR-74 (ND+LR-74) at 50 mg/L in drinking water.

Both plasma glucose and body weight were checked before administrationof the drug, and no differences were detected among the three diabetictreatment groups or among the three non-diabetic groups. All animalswere housed individually and were given free access to food and water.Glycemic control and body weights were monitored periodically. To limithyperglycemia and ensure that animals maintained body weight, diabeticanimals received 3 IU of ultralente insulin two to three times per week.The study was carried out over 32 weeks. Progression of renaldysfunction was assessed by measuring urinary albumin and plasmacreatinine concentrations according to known methods. Figarola et al.,Diabetologia 46: 1140-115, 2003.

Diabetic animals had higher glucose and HbAlc concentrations, and lowerbody weights than non-diabetic rats (P<0.001). Treatment with either L-9or LR-74 had no effect on hyperglycemia and body weight gains on eitherND or D rats. All diabetic animals initially comprised 9 animals in eachgroup. At the end of the study, the numbers were reduced in the diabeticcontrol (n=5), LR-9 (n=6) and LR-74 (n=6)-treated diabetic rats. Therewas no mortality observed in the non-diabetic groups.

Diabetes was associated with increased urinary albumin excretion andplasma creatinine concentration (P<0.001 vs. non-diabetic control) SeeTable V. Treatment of diabetic rats with either LR compound inhibitedthe rise in urinary albumin excretion, with about a 50% reduction inconcentration compared to untreated diabetic rats. The elevated plasmacreatinine concentrations observed in diabetic animals also weresignificantly decreased by almost 50% with treatment of either LR-9 orLR-74. Additionally, diabetic rats had higher kidney weights (measuredas a fraction of total body weight) compared with non-diabetic animals(P<0.05), indicating renal hypertrophy. Treatment of either LR compoundspartially attenuated these changes. See Table V. TABLE V Rat Physicaland Metabolic Parameters. Kidney/Body wt. Plasma Urinary Ratio^(a)Creatinine Albumin Group n (g/100 g) (mg/dl) mg/42 hr) ND 4 0.58 ± 0.020.45 ± 0.06  4.8 ± 0.9 ND + LR-9 4 0.51 ± 0.01 0.42 ± 0.02  4.8 ± 1.2ND + LR-74 4 0.52 ± 0.01 0.42 ± 0.03  4.6 ± 1.2 D 5 2.14 ± 0.27* 3.13 ±0.38* 32.8 ± 3.6* D + LR-9 6 1.58 ± 0.11** 1.79 ± 0.39** 18.0 ± 3.7**D + LR-74 6 1.52 ± 0.10** 1.64 ± 0.44** 14.3 ± 3.7***^(a)Ratio of left and right kidney weights to body weight.*P < 0.05 vs. ND;**P < 0.05 vs. D;***P < 0.01 vs. D.

Example 11 AGE Immunohistochemical Staining

At 32 weeks of the study, the rats were killed by over-anesthetizationwith isoflourane and cardiac puncture. Blood samples were collected fromeach animal and transferred accordingly into heparinized vacutainertubes and were later centrifuged for plasma isolation. Aliquots of theseplasma samples were stored at −70° C. until the time of analysis.Kidneys were removed immediately, decapsulated and rinsed in PBS buffer.Sections of the left kidneys were stored in 10% neutral bufferedformalin for subsequent microscopic examinations and AGEimmunohistochemistry. Sections of abdominal skin and tail of eachindividual rat were removed, rinsed in PBS buffer and stored at −70° C.for subsequent AGE quantification and crosslinking analyses.

Immunohistochemical staining for AGEs in rat kidney demonstrated thatthere was widespread staining for CML-AGE in the kidney glomeruli andcortical tubules in diabetic rats compared with the non-diabetic controlrats. Treatment with either LR compound clearly protected against theincrease in CML-AGE deposited in these regions, primarily in theglomeruli. See FIG. 18.

Example 12 AGE Formation in Collagen

Tail tendon collagen was isolated from each rat in Example 11 and thedegree of AGE formation in collagen was assessed by measurement offluorescent AGE after enzymatic digestion. Figarola et al., Diabetologia46: 1140-115, 2003. Skin collagen isolation and reduction was performedas described in Shaw et al., Methods Mol. Biol. 186: 129-137, 2002.Levels of AGEs/ALEs were normalized to the lysine content of thecollagen samples.

The levels of fluorescent AGEs in tail collagen increased aboutfive-fold in untreated diabetic rats compared to untreated non-diabeticanimals. See FIG. 19.

Ion-pair reversed-phase liquid chromatography/tandem mass spectroscopyanalysis was performed using an Agilent Technologies™ LC1100 seriessystem interfaced to a Micromass Quatro™ Ultima Triple Quadripole MassSpectrometer. HPLC separation was achieved using a Phenomenex SynergicHydro-RP 4 μM 80A 150×2.0 mm column preceded by a Phenomenex C18 guardcolumn. The column temperature was maintained at 25° C. and the flow was0.2 mL/minute. The isocratic mobile phase consisted of 10% acetonitrileand 0.1% heptafluorobutyric acid in water. Total run time was 12minutes; injection volume was 20 μL. The autoinjector temperature was 5°C. The electrospray ionization source of the mass spectrophotometer wasoperated in the positive ion mode with a cone gas flow of 190 L/hour anddesolvation gas flow of 550 L/hour. The capillary voltage was set to 2.7kV. Cone and collision cell voltages were optimized to 25 V and 13 eVfor CML, 33 V and 12 eV d₄CML, 24 V and 14 eV for CEL, 29 kV and 14 eVfor d₈CEL, and 29 kV and 16 eV for lysine, respectively. The sourcetemperature was 125° C. The desolvation temperature was increased to300° C., and the solvent delay program was used from 0 to 3 minutes and10 to 12 minutes. The fragmentation of these compounds can be inducedunder collision dissociation conditions and acidic mobile phase. Theprecursor->product ion combinations at m/z were 205.1->130.11 for CML,209.12->134.12 for d₄CML, 219.11->130.11 for CEL, 227.18->138.16 ford₈CEL, 147.15->84.21 for lysine and 151.27->88.33 for DL-d₄Lysine wereused in multiple reaction monitoring (MRM) mode to determine thesecompounds. MassLynx™ version 3.5 software was used for data acquisitionand processing.

All solutions of standards and internal standards were prepared inwater. Standard solution containing both CML and CEL were prepared atsix concentrations: 4, 10, 20, 40, 100 and 200 pmol/mL for CML and 2, 5,10, 20, 50 and 100 pmol/mL for CEL. Quality control solutions wereprepared at two concentrations: 7.5 and 150 pmol/mL for CML and 3.75 and75 pmol/mL for CEL. A stock solution of heavy-labeled internal standards(d₄CML and d₈CEL) was prepared at 800 pmol/mL. For generation of astandard curve, 130 μL of standard solution was freshly mixed with 10 μL0.1 M ammonium bicarbonate, 10 μL 0.05% HFBA and 10 μL internal standardstock solution to give a caliber. The calibrators then were assayed induplicate to establish the standard curves for CML and CEL. Thecalibration curve was plotted with the ratio of standard peak area tointernal standard peak area (Y) against the standard concentration (X).The standard curves, as determined by linear regression, displayed goodlinearity over the range tested (r²>0.99). For the treatment samples,each sample was further diluted 1:16 with water before mixing with theinternal standard solution. Then 130 μL of this diluted sample wasfreshly mixed with 10 μL 0.1 M ammonium bicarbonate, 10 μL 0.05% HFBAand 10 μL internal standard stock solution.

For lysine content determination, standard solutions of L-lysine wereprepared at 5 concentrations: 0.4, 0.8, 1.6, 3.2 and 6.4 nmol/mL.Quality control solutions were prepared at 2 concentrations: 0.6 and 5nmol. A stock solution of internal standards (DL-d₄Lysine) was prepareda 1 μg/mL and for the generation of standard curves, 100 μL of standardsolution was freshly mixed with 10 μL 0.05% HFBA and 20 μL internalstandard solution to give a calibrator. The calibrators then wereassayed in duplicate to establish the standard curves. The calibrationcurve was plotted with the ratio of standard peak area to internalstandard peak area (Y) against the standard concentration (X). Thestandard curves, as determined by linear regression, displayed goodlinearity over the range tested (r²>0.99). Each rehydrated collagensample was further diluted 1:3000 with water before mixing with internalstandard solution. Then 100 μL of diluted sample was freshly mixed with10 μL 0.05% HFBA and 20 μL internal standard stock solution.

Using the overall LC-ESI/MS/MS technique, the within-day coefficient ofvariation (CV) was less than 4.1% and less than 5.9% for CML and CEL,respectively. Between day CVs were less than 8.3% for CML and less than5.6% for CEL. Analysis of the AGE/ALE contents of skin collagen showedsignificant increase in both CML and CEL concentrations in untreateddiabetic animals versus untreated non-diabetic animals. LR-9 and LR-74treatment significantly limited the increase in both CML and CELconcentrations. See FIG. 20.

Example 13 Plasma Lipids

Diabetic rats showed elevated levels of plasma lipids compared withnon-diabetic rats. See FIG. 21. Plasma triglycerides increased to598±110 mg/dL in diabetic rats compared to 86±14 mg/dL in untreatednon-diabetic controls (P<0.001). Plasma cholesterol concentrationsshowed a similar increase in diabetic animals (61±7 mg/dL innon-diabetic vs. 136±13 mg/dL in diabetic rats) (P<0.001). Bothcompounds had no effect on lipid metabolism in non-diabetic animals.However, diabetic rats treated with either LR compounds showedsignificant reduction in both triglyceride and cholesterolconcentrations. LR-9 reduced plasma triglycerides and cholesterol by asmuch as 60% and 30%, respectively (means/SEM of 239±50 and 96±5 mg/dL,respectively). LR-74 treatment resulted in almost 70% reduction inplasma triglycerides (161±29 mg/dL) and approximately 30% decrease incholesterol levels (93±2 mg/dL) compared with untreated diabeticanimals. Plasma lipid hydroperoxide concentrations were approximatelyfive times higher in diabetic control rats compared with non-diabeticanimals (26.3±2.7 μM vs. 5.6±0.5 μM). See FIG. 21. Treatment with LR-9or LR-74 substantially reduced plasma lipid hydroperoxides in diabeticanimals by 35% and 45%, respectively. See FIG. 21.

Example 14 Effects of LR-9 and LR-74 on Body Weight and Glycemia inSTZ-Diabetic Rats

Rats were treated and divided into treatment groups as described inExample 10. Both plasma glucose and body weight were checked beforeadministration of the drug, and no differences were detected among thethree diabetic treatment groups or among the three non-diabetic groups.All animals were housed individually and were given free access to foodand water. Glycemic control and body weights were monitoredperiodically. To limit hyperglycemia and ensure that animals maintainedbody weight, diabetic animals received 3 IU of ultralente insulin two tothree times per week. The study was carried out over 32 weeks.

As described above, the diabetic animals had higher glucose and HbAlcconcentrations, and lower body weights than non-diabetic rats (P<0.001).Treatment with either L-9 or LR-74 had no effect on hyperglycemia andbody weight gains on either ND or D rats. All diabetic animals initiallycomprised 9 animals in each group. At the end of the study, the numberswere reduced in the diabetic control (n=5), LR-9 (n=6) and LR-74 (n=6)treated diabetic rats. See Table VI. There was no mortality observed inthe non-diabetic groups. See Examples 1 and 10. TABLE VI Body Weight andGlycemia in STZ-Diabetic Rats. Body Weight Plasma Glucose Group n (g)(mmol/l) HbA1c (%) ND^(a) 4 668.5 ± 32.8  8.5 ± 0.4 0.9 ± 0.1 ND + LR-94 681.0 ± 4.6  8.2 ± 0.5 0.9 ± 0.1 ND + LR-74 4 744.0 ± 25.8  6.8 ± 0.40.9 ± 0.1 D^(a) 5 250.8 ± 19.3* 26.5 ± 1.0* 2.1 ± 0.1* D + LR-9 6 288.0± 29.3* 27.1 ± 0.5* 2.0 ± 0.1* D + LR-74 6 314.7 ± 21.6* 26.9 ± 0.6* 2.1± 0.1*^(a)ND = non-diabetic; D = diabetic.*indicates P < 0.05 vs. ND rats.

Example 15 Nitrotyrosine Formation is Increased in Diabetic Rats

Formalin-fixed parafilm embedded kidney sections (2 μm thick) weremounted on slides and stained with polyclonal anti-nitrotyrosineantibodies according to previously known methods. See Figarola et al.,Diabetologia 46: 1140-1152. Nitrotyrosine formation, an index of proteinoxidative damage resulting from reactive nitrogen species, was enhancedin diabetic animals, specifically in the proximal tubule cells. See FIG.22. This increased staining was attenuated by treatment of either LRcompound.

Example 16 In Vitro Lipid Peroxidation Effects on Human Samples

Human LDL was isolated from plasma of healthy donors by single verticalspin centrifugation (see Chung et al., Methods Enzymol. 128: 181-209,1986) and used within 24-48 hours of preparation. LDL (50 μg/mL) wasincubated at 37° C. in 50 mM chelex-treated phosphate buffer, pH 7.4,alone or in the presence of 5 μM CuCl₂ or 5 μM CuCl₂ plus variousconcentrations of the LR compounds. After 5 hours of incubation,aliquots from each reaction mixture were removed for measurement ofthiobarbituric acid-reacting substances (TBARS) as described in Satoh,Clin. Chim. Acta 90: 37-43, 1978. Briefly, 250 μL of 20% trichloroaceticacid was added to 500 μL of sample aliquot, followed by 750 μL of 1%TBARS. The samples then were vortexed and incubated in a boiling waterbath for 10 minutes. Upon cooling, the samples were centrifuged for 5minutes at 5000 rpm. Absorbance of the supernatant was taken at 532 nm,and expressed as MDA equivalents using 1,1,3,3-tetramethoxypropane asstandards. For fatty acid oxidation studies, linoleic acid (5#mM) wasincubated alone or in the presence of 1 mM LR compound in 200 nMphosphate buffer, pH 7.4, for 7 days at 37° C. Aliquots from eachreaction mixture were withdrawn periodically for measurement of TBARS asdescribed above. Aminoquanidine (AG) and pyridoxamine (PM) were used at250 μM as comparative controls.

Results are shown in FIG. 23A, where values are provided as means±SD oftwo independent experiments (n=4 for each treatment). In separateexperiments, the time course for the oxidative modification fo LDL byCu⁺⁺ in the presence of 250 μM compound was followed for 5 hours andaliquots for each time interval were assayed for TBARS. See FIG. 23B.Values in FIG. 23B are means±SD of two independent experiments (n=4 pertreatment).

As shown in FIG. 23A, the LR compounds inhibited human low-densitylipoprotein (LDL) oxidation in a concentration-dependent manner betterthan AG and PM. The kinetics of Cu⁺⁺-mediated oxidation of LDL ischaracterized by two phases, a lag phase of about 2 hours and apropagation phase. The presence of either LR-74 or LR-90 extended thelag phase to such a degree that there was no observable propagationphase. See FIG. 23B. LR-9 significantly inhibited the rate of oxidationafter 2 hours compared to the control, while PM had no effect onmetal-catalyzed LDL oxidation.

In a kinetic study of the oxidation of linoleic acid (LA), the mainfatty acid in LDL, LR compounds prevented the formation of lipidperoxidation products, particularly MDA and related aldehydes. See FIG.24. Linoleic acid (5 mM) was incubated alone or in the presence of 1 mMLR compound or pyridoxamine (PM), as indicated, in 200 mM phosphatebuffer, pH 7.4, for 7 days at 37° C. Aliquots were withdrawnperiodically and assayed by the TBARS method as above. The MDAequivalent was estimated based on standards.

LA oxidation increased and reached its peak within 3 days of incubation,then gradually declined after that period. LR-9 and LR-90 totallyprevented LA oxidation throughout the 7-day incubation period. LR-74 didnot totally prevent the oxidation; it inhibited the maximum oxidationobserved at day 3. On the other hand, similar to observations with LDLoxidation, the AGE/ALE inhibitor PM had no effect on LA oxidation. SeeFIG. 24.

1. A method of lowering lipid levels in a mammal comprisingadministering an effective amount of a compound or a pharmaceuticallyacceptable salt of said compound to said mammal wherein said compound isselected from the group consisting of: LR-9[4-(2-napthylcarboxamido)phenoxyisobutyric acid]; LR-74[2-(8-quinolinoxy)propionic acid]; and LR-90 [methylenebis(4,4′-(2-chlorophenylureidophenoxyisobutyric acid)].
 2. The method ofclaim 1 wherein said compound is LR-9:[4-(2-napthylcarboxamido)phenoxyisobutyric acid].
 3. The method of claim1 wherein said compound is LR-74: [2-(8-quinolinoxy)propionic acid]. 4.The method of claim 1 wherein said compound is LR-90: [methylenebis(4,4′-(2-chlorophenylureidophenoxyisobutyric acid)].
 5. A method oftreating complications resulting from diabetes wherein saidcomplications result from elevated levels of lipids, said methodcomprising administering an effective amount of a compound or apharmaceutically acceptable salt of said compound to a mammal whereinsaid compound is selected from the group consisting of: LR-9[4-(2-napthylcarboxamido)phenoxyisobutyric acid]; LR-74[2-(8-quinolinoxy)propionic acid]; and LR-90 [methylenebis(4,4′-(2-chlorophenylureidophenoxyisobutyric acid)].
 6. The method ofclaim 5 wherein said compound is LR-9[4-(2-napthylcarboxamido)phenoxyisobutyric acid].
 7. The method of claim5 wherein said compound is LR-74 [2-(8 quinolinoxypropionic acid]. 8.The method of claim 5 wherein said compound is LR-90 [methylenebis(4,4′-(2-chlorophenylureidophenoxyisobutyric acid)].
 9. A method oftreating a patient with Menkes Disease, Wilson's Disease, or X-linkedCutis Laxa, wherein said method comprises administering an effectiveamount of a compound or a pharmaceutically acceptable salt of saidcompound to said patient, wherein said compound is selected from thegroup consisting of: LR-9 [4-(2-napthylcarboxamido)phenoxyisobutyricacid]; LR-74 [2-(8-quinolinoxy)propionic acid]; and LR-90 [methylenebis(4,4′-(2-chlorophenylureidophenoxyisobutyric acid)].